Anti-inflammatory pharmaceutical compositions for reducing inflammation and the treatment of prevention of gastric toxicity

ABSTRACT

The invention provides hops ( Humulis lupulus ) extracts or derivatives thereof for use in treating a patient prophylactically and/or therapeutically for ulcerogenic-type disorders of the stomach and/or intestines. The ulcerogenic disorders can be of the type chemically induced, environmentally-induced, infection-induced, and/or stress-induced. The invention also provides a pharmaceutical composition comprising an active amount of hops extracts or derivatives thereof, in combination with an analgesic compound and/or an anti-inflammatory compound. The invention further provides for use of hops extracts or derivatives thereof, significantly reducing and/or therapeutically treating ulcerogenic-type disorders of the stomach and/or intestines.

This application claims the benefit of priority of U.S. Provisionalapplication Ser. No. 60/472,460, filed May 22, 2003, provisionalapplication No. 60/420,383, filed Oct. 21, 2002, and provisionalapplication No. 60/450,237 filed Feb. 25, 2003; and is acontinuation-in-part of application Ser. No. 09/885,721 filed Jun. 20,2001, each of which the entire contents is incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention relates to pharmaceutical compositions containing hops(Humulus lupulus) extracts or derivatives thereof. The present inventionalso relates to methods of using compositions obtained or derived fromhops to treat, prevent or attenuate gastropathy, and particularly butnot exclusively that caused by non-steroidal anti-inflammatory drugs.

Prostaglandins (PGs) are ubiquitous hormones that function as bothparacrine and autocrine mediators to affect a myriad of physiologicalchanges in the immediate cellular environment. The varied physiologicaleffects of PGs include inflammatory reactions such as rheumatoidarthritis and osteoarthritis, blood pressure control, plateletaggregation, induction of labor and aggravation of pain and fever. Thediscovery 30 years ago that aspirin and other non-steroidal analgesicsinhibited PG production identified PG synthesis as a target for drugdevelopment. There are at least 16 different PGs in nine differentchemical classes, designated PGA to PGI. PGs are part of a larger familyof 20-carbon-containing compounds called eicosanoids; they includeprostacyclins, thromboxanes, and leukotrienes. The array of PGs producedvaries depending on the downstream enzymatic machinery present in aparticular cell type. For example, endothelial cells produce primarilyPGI₂, whereas platelets mainly produce TXA₂.

Arachidonic acid serves as the primary substrate for the biosynthesis ofall PGs. Cyclooxygenase (prostaglandin endoperoxide synthase, EC1.14.991, COX) catalyzes the rate-limiting step in the metabolism ofarachidonic acid to prostaglandin H₂ (PGH₂), which is furthermetabolized to various prostaglandins, prostacyclin and thromboxane A2(see FIG. 1). In the early 1990s, it was established that COX exists intwo isoforms, commonly referred to as COX-1 and COX-2. It wassubsequently determined that the COX-1 and COX-2 proteins are derivedfrom distinct genes that diverged well before birds and mammals. PGsgenerated via the COX-1 and COX-2 pathways are identical molecules andtherefore have identical biological effects. COX-1 and COX-2, however,may generate a unique pattern and variable amounts of eicosanoids;therefore, relative differences in the activation of these isozymes mayresult in quite dissimilar biological responses. Differences in thetissue distribution and regulation of COX-1 and COX-2 are now consideredcrucial for the beneficial as well as adverse effects of COX inhibitors.

The generally held concept (COX dogma) is that COX-1 is expressedconstitutively in most tissues whereas COX-2 is the inducible enzymetriggered by pro-inflammatory stimuli including mitogens, cytokines andbacterial lipopolysaccharide (LPS) in cells in vitro and in inflamedsites in vivo. Based primarily on such differences in expression, COX-1has been characterized as a housekeeping enzyme and is thought to beinvolved in maintaining physiological functions such as cytoprotectionof the gastric mucosa, regulation of renal blood flow, and control ofplatelet aggregation. COX-2 is considered to mainly mediateinflammation, although constitutive expression is found in brain, kidneyand the gastrointestinal tract.

Prostaglandins (PG) are believed to play an important role inmaintenance of human gastric mucosal homeostasis. Current dogma is thatCOX-1 is responsible for PG synthesis in normal gastric mucosa in orderto maintain mucosal homeostasis and that COX-2 is expressed by normalgastric mucosa at low levels, with induction of expression during ulcerhealing, following endotoxin exposure or cytokine stimulation. It nowappears that both COX-1 and COX-2 have important physiological roles inthe normal gastric mucosa.

Compounds that inhibit the production of PGs by COX have becomeimportant drugs in the control of pain and inflammation. Collectivelythese agents are known as non-steroidal anti-inflammatory drugs (NSAIDs)with their main indications being osteoarthritis and rheumatoidarthritis. However, the use of NSAIDs, and in particular aspirin, hasbeen extended to prophylaxis of cardiovascular disease. Over the lastdecade, considerable effort has been devoted to developing new moleculesthat are direct inhibitors of the enzymatic activity of COX-2, with theinference that these compounds would be less irritating to the stomachwith chronic use.

The major problem associated with ascertaining COX-2 selectivity (i.e.low gastric irritancy) is that differences in assay methodology can haveprofound effects on the results obtained. Depicted in Table 1 are thecategories of the numerous in vitro assays that have been developed fortesting and comparing the relative inhibitory activities of NSAID andnatural compounds against COX-1 and COX-2. These test systems can beclassified into three groups: (1) systems using animal enzymes, animalcells or cell lines, (2) assays using human cell lines, or humanplatelets and monocytes, and (3) currently evolving models using humancells that are representative of the target cells for theanti-inflammatory and adverse effects of NSAID and dietary supplements.Generally, models using human cell lines or human platelets andmonocytes are the current standard and validated target cell models havenot been forthcoming. A human gastric cell line capable of assessingpotential for gastric irritancy is a critical need.

The enzymes used can be of animal or human origin, they can be native orrecombinant, and they can be used either as purified enzymes, inmicrosomal preparations, or in whole-cell assays. Other system variablesinclude the source of arachidonic acid. PG synthesis can be measuredfrom endogenously released arachidonic acid or exogenously addedarachidonic acid. In the later case, different concentrations are usedin different laboratories.

An ideal assay for COX-2 selectivity would have the followingcharacteristics: (1) whole cells should be used that contain nativehuman enzymes under normal physiological control regarding expression;(2) the cells should also be target cells for the anti-inflammatory andadverse effects of the compounds; (3) COX-2 should be induced, therebysimulating an inflammatory process, rather than being constitutivelyexpressed; and (4) PG synthesis should be measured from arachidonic acidreleased from endogenous stores rather than from exogenously addedarachidonic acid. TABLE 1 Classification of test systems for in vitroassays assessing COX-2 selectivity of anti-inflammatory compounds† I.TEST SYSTEMS A. ANIMAL B. HUMAN C. TARGET Enzymes Enzymes Human GastricMucosa Cells Cells Cells Human Chondrocytes Cell lines Cell lines HumanSynoviocytes D. OTHER SYSTEM VARIABLES 1. Source of arachidonic acid -endogenous or exogenous; 2. Various expression systems for genereplication of COX-1 and COX-2; 3. The presence or absence of a COX-2inducing agent; 4. COX-2 inducing agents are administered at differentconcentrations and for different periods of time; 5. Duration ofincubation with the drug or with arachidonic acid; 6. Variation in theprotein concentration in the medium.†Adapted from Pairet, M. and van Ryn, J. (1998) Experimental models usedto investigate the differential inhibition of cyclooxygenase-1 andcyclooxygenase-2 by non-steroidal anti-inflammatory drugs. Inflamm. Res47, Supplement 2S93-S101 and incorporated herein by reference.

No laboratory has yet developed an ideal assay for COX-2 selectivity.The whole cell system most commonly used for prescription (Rx) and overthe counter (OTC) products is the human whole blood assay developed bythe William Harvey Institute [Warner, T. D. et al. (1999) Nonsteroiddrug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2are associated with human gastrointestinal toxicity: a full in vitroanalysis. Proc Natl Acad Sci USA 96,7563-7568]. To date, this assayformat has developed more data supporting clinical relevance than anyother. However, new research in the role of constitutive expression ofCOX-2 in normal gastric mucosa necessitates revisiting the relevance ofthe use of platelets to model COX-1 inhibition in the absence of COX-2.The extrapolation of gastrotoxicity from platelet studies is no longeron a sound molecular basis. The validation of a human gastric mucosalcell line for establishing the potential target tissue toxicity ofcyclooxygenase inhibitors represents a critical need for the developmentof safe and effective anti-inflammatory agents.

An ideal formulation for the treatment of inflammation would inhibit theinduction and activity of COX-2 without inhibiting the synthesis of PGE₂in gastric mucosal cells. However, conventional non-steroidalanti-inflammatory drugs lack the specificity of inhibiting COX-2 withoutaffecting gastric PGE₂ synthesis and are at risk to cause damages on thegastrointestinal system, when used for extended periods. Indeed, eventhe newly developed, anti-inflammatory drugs such as rofecoxib (Vioxx®,Merck & Co., Inc.) and celecoxib (Celebrex®, Pfizer, Inc.) produceuntoward gastric toxicity in the form of induced spontaneous bleedingand delay of gastric ulcer healing.

NSAID Toxicity

NSAIDs are known to cause serious health problems including gastricbleeding and kidney damage. In the United States, there are over 13million regular users of NSAIDs, 70 million NSAID prescriptions writtenevery year, and 30 billion over the counter NSAIDs tablets soldannually. NSAID-induced disease causes 103,000 hospitalizations per yearand an estimated 16,500 deaths annually. Twenty percent of all chronicNSAID users will develop a peptic ulcer. NSAID users have a greaterrisk—three to four times higher—to upper gastrointestinal bleeding,perforation, or both. Eighty-one percent of patients hospitalized withserious NSAID-induced complications had no previous gastrointestinalsymptoms. People over 60 years of age have a significantly higherprobability of experiencing complications associated with NSAID use.Moreover, 21% of all adverse drug reaction in the United States are dueto NSAID use.

The new selective COX-2 inhibitors such as celecoxib and rofecoxib havebeen shown to offer a safer alternative to most NSAIDs. However recentstudies indicate that selective COX-2 inhibitors do not completelyeliminate gastrointestinal toxicity. In fact in cases of inflammation orulceration of the gastrointestinal tract, prescription COX-2 inhibitorsmay delay ulcer healing.

Thus, it would be useful to identify a natural formulation of compoundsthat would specifically inhibit or prevent the synthesis ofprostaglandins by COX-2 with little or no effect on synthesis of PGE₂ inthe gastric mucosa. Such a formulation, which would be useful forpreserving the health of joint tissues, for treating arthritis or otherinflammatory conditions, has not previously been discovered. The term“specific or selective COX-2 inhibitor” was coined to embrace compoundsor mixtures of compounds that selectively inhibit COX-2 over COX-1.However, while the implication is that such a calculated selectivitywill result in lower gastric irritancy, unless the test materials areevaluated in gastric cells, the term “selective COX-2 inhibitor” doesnot carry assurance of safety to gastrointestinal cells. Only testing ofcompound action in target tissues, inflammatory cells and gastricmucosal cells, will identify those agents with low potential for stomachirritation.

Therefore, it would be useful to identify a composition that wouldspecifically inhibit or prevent the expression of COX-2 enzymaticactivity in inflammatory cells, while having little or no effect on PGE₂synthesis in gastric mucosal cells so that these formulations could beused with no gastrointestinal upset. Furthermore, such formulationsshould allow for healing of pre-existing ulcerative conditions in thestomach. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The invention provides compositions and methods using fractions isolatedor derived from hops to ameliorate, inhibit or prevent gastropathyand/or gastroenteropathy associated with a gastriointestinal irritant.The compositions can be combined with other components to enhancedesirable effects and inhibit undesirable effects of a second component,for example, non-steroidal anti-inflammatory drug, spices, or othergastrointestinal irritants. The invention also provides methods forreducing gastroenteropathy or gastric toxicity, includingulcerogenic-type disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the induction of cyclooxygenase-2 and the metabolism ofarachidonic acid to prostaglandins and other eicosanoids by thecyclooxygenase enzymes. The action of non-steroidal anti-inflammatoryagents is through direct inhibition of the cyclooxygenase enzymes.

FIG. 2 shows an outline of fractions and compounds that can be obtainedfrom hops.

FIG. 3 illustrates exemplary fractions isolated or derived from hops.FIG. 3A shows the alpha-acid genus (AA) and representative specieshumulone (R═—CH₂CH(CH₃)₂), cohumulone (R═, —CH(CH₃)₂), and adhumulone(R═CH(CH₃)CH₂CH₃); FIG. 3B shows the isoalpha acid genus (IAA) andrepresentative species isohumulone (R═—CH₂CH(CH₃)₂), isocohumulone (R═,—CH(CH₃)₂), and isoadhumulone (R═—CH(CH₃)CH₂CH₃); FIG. 3C shows thereduced isomerized isoalpha acid genus (RIAA) and representative speciesdihydro-isohumulone (R═CH₂CH(CH₃)₂) dihydro-isocohumulone (R═,—CH(CH₃)₂), and dihydro-adhumulone (R═CH(CH₃)CH₂CH₃); FIG. 3D shows thetetra-hydroisoalpha acid genus (THIAA) and representative speciestetra-hydro-isohumulone (R═CH₂CH(CH₃)₂), tetra-hydro-isocohumulone ((R═,—CH(CH₃)₂), and tetra-hydro-adhumulone (R═CH(CH₃)CH₂CH₃); FIG. 3E showsand the hexa-hydroisoalpha acid (HHIAA) genus with representativespecies hexa-hydro-isohumulone (R═CH₂CH(CH₃)₂) hexa-hydro-isocohumulone(R═, —CH(CH₃)₂), and hexa-hydro-adhumulone (R═—CH(CH₃)CH₂CH₃).

FIG. 4 depicts a representative immunoblots demonstrating constitutiveCOX-1 and COX-2 expression in AGS human gastric mucosal cells. The AGShuman gastric cell line was cultured in 6-well plates at 37° C. with 5%CO₂ in a humidified incubator for 24 hours. Cells were lysed on ice inlysis buffer and protein concentration determined. Fifty μg of celllysate were solubilized, fractionated on a 10% polyacrylamide gelcontaining sodium dodecylsulfate (SDS), and transferred onto anitrocellulose membrane. The membranes were incubated in a blockingbuffer and then incubated with the respective primary antibody for 1 hat room temperature. Following primary antibody incubation, the blotswere washed three times with Tris-buffered saline and then incubatedwith the secondary antibody for 1 h. Protein bands were visualized usingenhanced chemiluminescence.

FIG. 5 depicts a comparison of Log IC₅₀ ratios (AGS/WHMA COX-2). Valuesto the right of 0 indicate decreasing probability of gastrointestinaleffects, while values to the left of 0 indicate increasing probabilityof gastrointestinal effects.

FIG. 6 illustrates percent inhibition of PGE₂ biosynthesis in AGSgastric mucosal cells by representative Genus A reduced isomerized alphaacid (RIAA) species. FIG. 6A shows ibuprofen and RIAA:ibuprofencombinations at 5 (gray bars) and 0.5 (white bars) μg test material/mL.FIG. 6B shows aspirin and RIAA:aspirin combinations at 5 (striped bars)and 0.5 (white bars) μg test material/mL.

FIG. 7 illustrates percent inhibition of PGE₂ biosynthesis in AGSgastric mucosal cells by representative Genus B tetra-hydroisoalpha acid(THIAA) species and NSAIDS. FIG. 7A shows ibuprofen and THIAA:ibuprofencombinations at 5 (gray bars) and 0.5 (white bars) μg test material/mL.FIG. 7B shows aspirin and THIAA:aspirin combinations at 5 (striped bars)and 0.5 (white bars) μg test material/mL.

FIG. 8 depicts a comparison of Log IC₅₀ ratios (AGS/COX-2). FIG. 8 Ashows ibuprofen, RIAA and a 1:1 combination of RIAA:ibuprofen. FIG. 8Bshows aspirin, RIAA and a 1:1 combination of RIAA:aspirin. Values to theright of 0 indicate decreasing probability of gastrointestinal effects,while values to the left of 0 indicate increasing probability ofgastrointestinal effects.

FIG. 9 depicts a comparison of Log IC₅₀ ratios (AGS/COX-2). FIG. 9Ashows ibuprofen, THIAA, a 1:100 combination of THIAA:ibuprofen and a 1:1combination of THIAA:ibuprofen. FIG. 9B shows aspirin, THIAA, a 1:100combination of THIAA:aprin, and a 1:1 combination of THIAA:aspirin.Values to the right of 0 indicate decreasing probability ofgastrointestinal effects, while values to the left of 0 indicateincreasing probability of gastrointestinal effects.

FIG. 10 shows a schematic of clinical trial comparing a compositioncomprising hops extract and anti-inflammatory medications. Twenty-sixsubjects were screened, 23 subjects entered the trial, and 21 subjectscompleted the trial. Of those who did not complete the trial, 3 withdrewfor personal reasons, 2 from ARM 2 and 1 from ARM 1. Visits occurred asfollows: visit 1 (VI), day 0 of treatment A; V2, 7±1 days of treatmentA; V3, 14±1 days of treatment A; Washout, 21±1 days; V4, day 0 oftreatment B; V5, 7±1 days of treatment B; V6, 14±1 days after treatmentB.

FIG. 11 shows individual calprotectin levels for the subjects whocompleted the clinical trial (N=21). Baseline 1 is the average V1 valueand Baseline 2 is the value after the 21-day wash-out. The 7 day and 14day data for IP2003-001 CT and naproxen are shown. (Reference range<50μg/g stool).

FIG. 12 shows the pooled mean (±sd) calprotectin for the subjects whocompleted the clinical trial (N=21). Baseline 1 is the average V1 valueand Baseline 2 is the value after the 21-day wash-out. No significantdifference was seen between Baseline 1 and Baseline 2. No significantdifference was seen between Baseline 1 and either 7-day or 14-dayIP2003-001 CT treatment as well. However, both 7-day and 14-day naproxenvalues were significantly elevated from Baseline 1 (p<0.05). (Referencerange<50 μg/g stool).

FIG. 13 shows individual calprotectin for the subjects who had baselinevalues within the reference range of <50 μg/g stool (N=15). Baseline 1is the average V1 value and Baseline 2 is the value after the 21-daywash-out. The 7 day and 14 day data for IP2003-001 CT and naproxen arealso shown.

FIG. 14 shows mean (±sd) calprotectin levels for the subjects who hadbaseline values within the reference range of <50 μg/g stool (N=15).Baseline 1 is the average V1 value and Baseline 2 is the value after the21-day wash-out. The 7 day and 14 day data for IP2003-001CT and naproxenare also shown.

FIG. 15 shows individual calprotectin for the subjects who had baselinevalues above the reference range, or >50 μg/g stool (N=6). Baseline 1 isthe average of both V1 values and Baseline 2 is the value after the21-day wash-out. The 7 day and 14 day data for IP2003-001CT and naproxenare shown.

FIG. 16 shows mean fecal calprotectin for the subjects who had baselinevalues above the reference range, or >50 μg/g stool (N=6). Baseline 1 isthe average of both V1 values and Baseline 2 is the value after the21-day wash-out. The 7 day and 14 day data for IP2003-001 CT andnaproxen are shown.

FIG. 17 shows pooled calprotectin data (mean±sem) for the subjects onthe clinical trial. Baseline 1 is the average of all V1 baseline values.Baseline 2 is the average of all wash-out values. The 7 day and 14 daydata for IP2003-001CT and naproxen were pooled for this analysis,respectively. No significant difference was observed between Baseline 1,Baseline 2, or IP2003-001; however, naproxen calprotectin levels weresignificantly elevated after treatment (p<0.05; Wilcoxon/Kruskal-Wallisranked sums).

FIG. 18 shows median inhibitory concentrations for PGE₂ synthesis ofUltraInflamX™ components in the AGS cell model.

FIG. 19 shows expected and observed median inhibitory concentrations forPGE₂ synthesis of UltraInflamX™ components in the AGS cell model. IC₅₀values were computed from the average of three independent assays; AGScells were plated and allowed to reach 80% confluence. Cells were washedand test material was added 60 minutes prior to treatment with A23187.Thirty minutes later, media was removed for PGE₂ determination. Observedand expected IC₅₀ value for composite is based on weight of whole sample(nine components) and the observed and estimated IC₅₀ value forcomposite actives is based upon the percent weight of the four activesin the composite sample (45.3%).

FIG. 20 shows COX-2 selectivity of prescription (Rx), over the counter(OTC), and dietary supplement health and education act (DSHEA)compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for modulatingthe gastrointestinal toxicity of non-steroidal anti-inflammatory drugs(NSAIDs). In particular, the invention provides a supragenus ofcomponents isolated or derived from hops (Humulus lupulus) that have amodulating effect on the gastrointestinal toxicity of NSAIDs.Specifically, hops derivatives decrease gastric PGE₂ inhibition ofNSAIDs, producing a more favorable therapeutic index for NSAIDs. Thecompositions and methods of the invention are advantageous in that thehops derivatives increase the effect of NSAIDs on inflammatory, targetcells, thereby further increasing the therapeutic index for NSAIDs. Hopsderivatives can also be administered to treat, ameliorate or preventgastric ulcers induced by the administration of NSAIDs.

The invention provides hops (Humulus lupulus) extracts or derivativesthereof for use in treating a patient prophylactically and/ortherapeutically for ulcerogenic-type disorders of the stomach and/orintestines. The ulcerogenic disorders can be of the type chemicallyinduced and/or stress-induced. The invention also provides apharmaceutical composition comprising an active amount of hops extractsor derivatives thereof, in combination with an analgesic compound and/oran anti-inflammatory compound. The invention further provides for use ofhops extracts or derivatives thereof, significantly reducing and/ortherapeutically treating ulcerogenic-type disorders of the stomachand/or intestines.

As disclosed herein, hops derivatives are effective in decreasing theinhibiting effects of NSAIDs on PGE₂ synthesis in gastric mucosal cells,while maintaining or enhancing the PGE₂ inhibitory effect ininflammatory cells. In addition, it has been found that chemicallyinduced ulceration, produced by analgesic and/or anti-inflammatory drugssuch as ibuprofen, aspirin and indomethacin, or other chemical agents,is significantly reduced when these drugs are administered inconjunction with hop derivatives. Furthermore, it has been found thatthe toxicity of the organic acids, for example, salicylic acid and otheranalgesic and/or anti-inflammatory drugs, is reduced to a significantextent when administered in conjunction with hops derivatives. Thecompositions and methods of the invention are advantageous in that theintrinsic beneficial activity of the analgesic and/or anti-inflammatorydrugs is not detrimentally influenced by their use in conjunction withhops derivatives.

As further disclosed herein, it has also been found that hopsderivatives possess analgesic activity and that the combined use of hopsderivatives with analgesic drugs results in an increased analgesiceffect. It has furthermore been found that pharmaceutical preparationscomprising hops derivatives are also significantly effective in theprevention or reduction of stress induced gastro-intestinal ulcerations.Additionally, it has also been found that hops derivatives are effectivein the healing of ulcers. It will also be understood that, whenever hopsderivatives are used for the treatment of ulcers, the aforesaidanalgesic activity of hops derivatives may in addition be beneficial inrelieving the pain associated with ulcers.

The acute toxicity of hops derivatives is very low. Therefore,relatively high doses of hops derivatives can be used, if desired,without toxic effects due to the hops. Toxic doses are considerablyhigher than the therapeutic doses contemplated in accordance with thepresent invention.

The invention provides hops extracts or derivatives thereof for use intreating a patient prophylactically and/or therapeutically forulcerogenic-type disorders of the stomach and/or intestines. Theulcerogenic disorders can be of the type chemically induced and/orstress-induced. The invention also provides a pharmaceutical compositioncomprising an active amount of hops extracts or derivatives thereof, incombination with an analgesic compound and/or an anti-inflammatorycompound. The invention further provides for use of hops extracts orderivatives thereof, significantly reducing and/or therapeuticallytreating ulcerogenic-type disorders of the stomach and/or intestines.

As used herein, the term “dietary supplement” refers to compositionsconsumed to affect structural or functional changes in physiology. Theterm “therapeutic composition” refers to compounds administered to treator prevent a disease or to ameliorate a sign or symptom associated witha disease.

As used herein, the term “effective amount” means an amount necessary toachieve a selected result. Such an amount can be readily determinedwithout undue experimentation by a person of ordinary skill in the art.

As used herein, the term “substantial” means being largely but notwholly that which is specified.

As used herein, the term “COX inhibitor” refers to a composition ofcompounds that is capable of inhibiting the activity or expression ofCOX-2 enzymes or is capable of inhibiting or reducing the severity,including pain and swelling, of a severe inflammatory response.

As used herein, the terms “derivatives” or a matter “derived” refer to achemical substance related structurally to another substance andtheoretically obtainable from it, that is, a substance that can be madefrom another substance. Derivatives can include compounds obtained via achemical reaction. Methods of making derivatives of compounds are wellknown to those skilled in the art.

As used herein, the term “inflammatory cell” refers to those cellularmembers of the immune system, for example B and T lymphocytes,neutrophils or macrophages, involved in synthesis of prostaglandins inresponse to inflammatory signals such as interleukins, tumor necrosisfactor, bradykinin, histamine or bacterial-derived components.

As used herein, the term “target cells” refers to that cell populationin which the inhibition of PGE₂ or other prostaglandin synthesis isdesired, such as inflammatory cells or tumor cells. Alternatively,“non-target cells” refers to that cell population in which theinhibition of PGE₂ or other prostaglandin synthesis is not desired, suchas the gastric mucosal, neural or renal cells.

As used herein, the term “hop extract” refers to the solid materialresulting from (1) exposing a hops plant product to a solvent, (2)separating the solvent from the hops plant products, and (3) eliminatingthe solvent.

As used herein, the term “solvent” refers to a liquid of aqueous ororganic nature possessing the necessary characteristics to extract solidmaterial from the hop plant product. Examples of solvents would include,but are not limited to, water, steam, superheated water, methanol,ethanol, hexane, chloroform, methylene chloride, liquid supercriticalCO₂, liquid N₂, or combinations of such materials.

As used herein, the term “CO₂ extract” refers to the solid materialresulting from exposing a hops plant product to a liquid orsupercritical CO₂ preparation followed by the removing of the CO₂.

As used herein, the term “spent hops” refers to the solid andhydrophilic residue from extract of hops.

As used herein, the term “alpha acid” refers to compounds collectivelyknown as humulones and can be isolated from hops plant productsincluding, among others, humulone, cohumulone, adhumulone, hulupone, andisoprehumulone.

As used herein, the term “isoalpha acid” refers to compounds isolatedfrom hops plant products and which subsequently have been isomerized.The isomerization of alpha acids can occur thermally, such as boiling.Examples of isoalpha acids include, but are not limited to, isohumulone,isocohumulone, and isoadhumulone.

As used herein, the term “reduced isoalpha acid” refers to alpha acidsisolated from hops plant product and which subsequently have beenisomerized and reduced, including cis and trans forms. Examples ofreduced isoalpha acids (RIAA) include, but are not limited to,dihydro-isohumulone, dihydro-isocohumulone, and dihydro-adhumulone.

As used herein, the term “tetra-hydroisoalpha acid” refers to a certainclass of reduced isoalpha acid. Examples of tetra-hydroisoalpha acid(THLAA) include, but are not limited to, tetra-hydro-isohumulone,tetra-hydro-isocohumulone and tetra-hydro-adhumulone.

As used herein, the term “hexa-hydroisoalpha acid” refers to a certainclass of reduced isoalpha acid. Examples of hexa-hydroisoalpha acids(HHIAA) include, but are not limited to, hexa-hydro-isohumulone,hexa-hydro-isocohumulone and hexa-hydro-adhumulone.

As used herein, the term “beta-acid fraction” refers to compoundscollectively known as lupulones including, among others, lupulone,colupulone, adlupulone, tetrahydroisohumulone, and hexahydrocolupulone.

As used herein, the term “essential oil fraction” refers to a complexmixture of components including, among others, myrcene, humulene,beta-caryophyllene, undecane-2-on, and 2-methyl-but-3-en-ol.

As used herein, “conjugates” of compounds means compounds covalentlybound or conjugated to a member selected from the group consisting ofmono- or di-saccharides, amino acids, sulfates, succinate, acetate, andglutathione. The mono- or di-saccharide can be a member selected fromthe group consisting of glucose, mannose, ribose, galactose, rhamnose,arabinose, maltose, and fructose.

The invention relates to using hops extracts to reduce toxicityassociated with the administration of NSAIDs. Hop extraction in one formor another goes back over 150 years to the early nineteenth century whenextraction in water and ethanol was first attempted. Even today, anethanol extract is available in Europe, but by far the predominantextracts are organic solvent extracts (for example, hexane) and CO₂extracts (supercritical and liquid). CO₂ (typically at 60 bars pressureand 50 to 10° C.) is in a liquid state and is a relatively mild,non-polar solvent highly specific for hop soft resins and oils. Beyondthe critical point, typically at 300 bars pressure and 60° C., CO₂ hasthe properties of both a gas and a liquid and is a much strongersolvent. The composition of the various extracts is compared in Table 2.

At its simplest, hop extraction involves milling, pelleting andre-milling the hops to spread the lupulin, passing a solvent through apacked column to collect the resin components and finally, removal ofthe solvent to yield a whole or “pure” resin extract. TABLE 2 Hopextracts (Percent w/w) Organic Super-Critical Component Hops Solvent CO₂Liquid CO₂ Total resins 12-20 15-60  75-90 70-95 Alpha-acids  2-12 8-4527-55 30-60 Beta-acids  2-10 8-20 23-33 15-45 Essential oils 0.5-1.50-5  1-5  2-10 Hard resins 2-4 2-10  5-11 None Tannins  4-10 0.5-5  0.1-5   None Waxes 1-5 1-20  4-13  0-10 Water  8-12 1-15 1-7 1-5

The main organic extractants are strong solvents and in addition tovirtually all the lupulin components, they extract plant pigments,cuticular waxes, water and water-soluble materials.

Supercritical CO₂ is more selective than the organic solvents andextracts less of the tannins and waxes and less water and hencewater-soluble components. It does extract some of the plant pigmentslike chlorophyll but rather less than the organic solvents do. LiquidCO₂ is the most selective solvent used commercially for hops and henceproduces the most pure whole resin and oil extract. It extracts hardlythe hard resins or tannins, much lower levels of plant waxes, no plantpigments and less water and water-soluble materials.

As a consequence of this selectivity and the milder solvent properties,the absolute yield of liquid CO₂, extract per unit weight of hops isless than when using the other mentioned solvents. Additionally, theyield of alpha acids with liquid CO₂ (89-93%) is lower than that ofsupercritical CO₂ (91-94%) or the organic solvents (93-96%). Followingextraction, there is the process of solvent removal, which for organicsolvents involves heating to cause volatilization. Despite this, traceamounts of solvent do remain in the extract. The removal of CO₂,however, simply involves a release of pressure to volatize the CO₂.

As shown in FIG. 3, hops CO₂ extracts can be fractionated intocomponents, including hops oils, beta acids, and alpha acids. Hops oilsinclude, but are not limited to, humulene, beta-caryophyllene, mycrene,famescene, gamma-cadinene, alpha-selinene, and alpha-cadinene. Betaacids include, but are not limited to, lupulone, colupulone, adlupulone,tetrahydroisohumulone, and hexahydrocolupulone, collectively known aslupulones. Beta acids can be isomerized and reduced. Beta acids arereduced to give tetra-beta acids. Alpha acids include, but are notlimited to, humulone, cohumulone, adhumulone, hulupone, andisoprehumulone. Alpha acids can be isomerized to give isoalpha acids.Iso-alpha acids can be reduced to give reduced-isoalpha acids,tetra-hydroisoalpha acids, and hexa-hydroisoalpha acids.

The identification of humulone from hops extract as an inhibitor of boneresorption is reported in To be et al. (Biosci. Biotech. Biochem61(1):158-159 (1997)). Later studies by the same group characterized themechanism of action of humulone as inhibition of COX-2 genetranscription following TNFalpha stimulation of MC3T3, E1 cells(Yamamoto, FEBS Letters 465:103-106 (2000)). It was concluded that theaction of humulone (also humulon) was similar to that ofglucocorticoids, but that humulone did not function through theglucocorticoid receptor. While these results establish that humuloneinhibits PGE₂ synthesis in MC3T3 cells (osteoblasts) at the gene level,one skilled in the art would not assume that these results wouldnecessarily occur in immune inflammatory cells or other cell lines. Asdisclosed herein, hops compounds and derivatives exhibit a high degreeof tissue selectivity in target and non-target cells. Furthermore, thehops derivatives described in the present invention are structurallydistinct from the alpha acid humulone.

The invention provides compositions containing at least one fractionisolated or derived from hops (Humulus lupulus). Examples of fractionsisolated or derived from hops are alpha acids, isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, and spent hops. Fractions isolated or derived from hops,include, but are not limited to, cohumulone, adhumulone, isohumulone,isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone. Preferred compoundscan also bear substituents, such as halogens, ethers, and esters.

Compounds of the fractions isolated or derived from hops can berepresented by a supragenus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and p orbital, with the proviso that if one of R, T, X, orZ is a p orbital, then the adjacent R, T, X, or Z is also a p orbital,thereby forming a double bond.

In another embodiment, compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃. ExemplaryGenus A structures include isoalpha acids such as isohumulone,isocohumulone, isoadhumulone, and the like, and reduced isoalpha acidssuch as dihydro-isohumulone, dihydro-isocohumulone, dihydroadhumulone,and ether or ester conjugates or halogenated modifications of the doublebond.

In yet another embodiment, compounds of the fractions isolated orderived from hops can be represented by a genus below:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃. ExemplaryGenus B structures include tetra-hydroisoalpha acids such astetra-hydro-isohumulone, tetra-hydro-isocohymulone andtetra-hydro-adhumulone, and the like, and hexa-hydroisoalpha acids suchas hexa-hydro-isohumulone, hexa-hydro-isocohumulone andhexa-hydro-adhumulone, and ether or ester conjugates.

As shown in FIG. 3, examples of compounds of an ingredient isolated orderived from hops, include, but are not limited to, humulone,cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. The preferred compounds can bear substituents, asshown in the formula above.

Hops derivatives are known compounds occurring naturally in plants andfound in food products and beverages. They may be prepared by any of theextraction and processing methods known in the art. Hops derivatives canbe prepared directly from plant material in any known manner. The hopsderivatives may be purified by methods known in the art, for example, byrecrystallization from aqueous organic solvents such as aqueousalcohols. Synthetic modifications of hops derivatives may be preparedaccording to methods known in the pharmaceutical art of drugmodification.

The invention also provides compositions containing an analgesic and/orinflammatory compound or drug, for example, an NSAID, and a fraction orcompounds isolated or derived from hops. For example, the inventionprovides compositions containing a fraction or compounds isolated orderived from hops, as disclosed herein, and one or more analgesic and/orinflammatory compounds or drugs such as NSAIDs. In a particularembodiment, the invention provides a composition comprising an isoalphaacid or reduced isoalpha acid isolated from hops and a non-steroidalanti-inflammatory drug. The isoalpha acid can be selected fromisohumulone, isocohumulone, and isoadhumulone. In another embodiment ofthe invention, the reduced isoalpha acid can be selected fromdihydro-isohumulone, dihydro-isocohumulone, and dihydro-adhumulone.

Exemplary analgesic and/or anti-inflammatory compounds or drugs include,but are not limited to, the following substances: the salicylatesaspirin, salicylic acid, methyl salicylate, difulunisal, salsalate,olsalazine, and sulfasalazine; para-aminophenol derivatives acetanilide,acetaminophen, and phenacetin; the fenamates mefenamic acid,meclofenamate and sodium meclofenamate; the heteroaryl acetic acidderivatives tolmetin, ketorolac and diclofenac; the propionic acidderivatives ibuprofen, naproxen, sodium daproxen, fenoprofen,ketoprofen, flurbioprofen/flurbiprofen, and oxaprozin; the enolic acidsrepresented by oxicam derivatives piroxicam, meloxicam, tenoxicam,ampiroxicam, droxicam and pivoxicam; the pyrazolon derivativesphenylbutazone, oxyphenbutazone, anitpyrine, aminopyrine and dipyrone;the coxibs celecoxib, and rofecoxib; nabumetone; apazone; nimensulide;indomethacin; sulindac; etodolac; diflunisal, isobutylphenyl propionicacid, and any other substances used in the treatment of pain andinflammatory conditions that cause or promote gastro-intestinal damage.As used herein, a non-aspirin, non-steroidal anti-inflammatory compoundspecifically excludes aspirin, acetylsalicylic acid.

Also in accordance with the present invention there are providedpharmaceutical compositions comprising an effective amount of hopsderivatives optionally in combination with a pharmaceutical diluent oradjuvant. Further in accordance with the present invention, there areprovided pharmaceutical compositions comprising an effective amount ofhops derivatives in combination with one or more analgesic and/oranti-inflammatory compound(s) in an effective and tolerated amount andconcentration.

The invention additionally provides pharmaceutical compositionscomprising an effective amount of hops derivatives in combination withone or more compatible compound(s) effective in the treatment of stressconditions. Such compositions can be used, for example, for thetreatment of ulcers, and to reduce the formation of gastro-intestinalulcers whether chemically or stress-induced.

Dosage

Further in accordance with the present invention there are providedpharmaceutical formulations of oral dosage forms comprising an effectiveamount of hops derivatives for release of the active ingredient at adesired site in the gastro-intestinal tract, for instance either in thestomach and/or duodenum according to known formulation techniques, e.g.slow releasing tablets. Still further in accordance with the invention,there are provided pharmaceutical compositions comprising an effectivetolerated amount of hops derivatives and a known compound effective inpreventing ulcer formation and/or a known compound effective inthereapeutically treating ulcers and/or a known compound(s) effective inrelieving the symptoms associated with ulcers, such as an antacid, e.g.aluminum hydroxide. Due to its low toxicity, high dosages of hopsderivatives can be employed to produce useful results, depending uponthe particular effect that is desired.

Hops derivatives are particularly suitable for oral administration.Therefore, hops derivatives can be formulated for oral use, namely:tablets, coated tablets, dragees, capsules, powders, granulates andsoluble tablets, and liquid forms, for example, suspensions, dispersionsor solutions, optionally together with an additional active ingredient,such as one or more analgesic and/or anti-inflammatory compound(s).

The invention extends to a method of preparing such pharmaceuticalcompositions as described herein and compositions when so prepared. Thecompositions may be manufactured by a method which comprises mixing hopsderivatives with a pharmaceutically acceptable carrier or auxiliary, andoptionally with an analgesic and/or anti-inflammatory substance and/oranother compound(s). Methods for preparing a pharmaceutical compositionare well known to those skilled in the art (see, for example, Genarro,ed., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,Easton, Pa. (1990)).

The selected dosage level will depend upon the activity of theparticular composition, the route of administration, the severity of thecondition being treated or prevented, and the condition and priormedical history of the patient being treated. However, it is within theskill of the art to start doses of the composition at levels lower thanrequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. If desired,the effective daily dose may be divided into multiple doses for purposesof administration, for example, two to four separate doses per day. Itwill be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including bodyweight, general health, diet, time and route of administration,combination with other compositions and the severity of the particularcondition being treated or prevented. The efficacious dose may beadministered prior to, with, or subsequent to the NSAID that causesgastropathy.

The invention provides methods that include delivering an effectiveamount of hops fractions, hops compounds, or hops derivatives alone orin combination with one or more NSAID. For example, a daily dose ofcompositions of the invention can be formulated to deliver about 0.5 toabout 10,000 mg of a hops fraction, for example, alpha acid, isoalphaacid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, spent hops, or other hops fractions,per day. In particular, an effective daily dose of compositions can beformulated to deliver about 50 to about 7500 mg of hops fraction, forexample, alpha acids, isoalpha acid, reduced isoalpha acid,tetra-hydroisoalpha acid, hexa-hydroisoalpha acid, beta acid, spenthops, or other hops fractions, per day. For example, an effective dailydose of compositions can be formulated to deliver about 100 mg to about5000 mg, about 200 mg to about 3000 mg, about 300 mg to about 2000 mg,about 500 to about 1000 mg of hops fraction per day. In one embodiment,the effective daily dose is administered once or twice a day. A certainembodiment provides a composition comprising about 0.5 to about 500 mgof isoalpha acid or reduced isoalpha acid, for example, about 50 toabout 300 mg or about 100 to about 200 mg of isoalpha acid or reducedisoalpha acid per day. In another embodiment, the invention provides acomposition comprising about 10 to about 3000 mg of reduced isoalphaacid, tetra-hydroisoalpha acid, or hexa-hydroisoalpha acid per day, forexample, about 50 to about 2000 mg, about 100 to about 1000 mg, about200 to about 750 mg, or about 250 to about 500 mg of reduced isoalphaacid, tetra-hydroisoalpha acid, or hexa-hydroisoalpha acid per day. Yetanother certain embodiment provides a composition comprising about 50 toabout 7500 mg of spent hops per day, for example, about 100 to about6000 mg, about 200 to about 5000 mg, about 300 to about 3000 mg, about500 to about 2000 mg, or about 1000 to about 1500 mg of spent hops perday.

A composition of embodiments for topical application can contain about0.001 to about 10 weight percent, for example, about 0.01 to about 5weight percent, or about 0.1 to about 1 weight percent, of a hopsderivative. Such compositions can produce serum concentrations in therange of about 0.0001 to about 10 μM, for example, about 0.001 to about5 μM, about 0.01 to 1 μM, or about 0.1 to about 0.5 μM of a fractionisolated or derived from hops or conjugate thereof.

Formulations

Compositions of the invention can be administered in the form of adietary supplement or therapeutic composition. The compositions may beadministered orally, topically, transdermally, transmucosally,parenterally, and the like, in appropriate dosage units, as desired.Compositions for dietary application may include various additives suchas other natural components of intermediary metabolism, vitamins andminerals, as well as inert ingredients such as talc and magnesiumstearate that are standard excipients in the manufacture of tablets andcapsules. For example, one embodiment comprises active ingredients ofcompositions of the invention in combination with glucosamine orchondrotin sulfate.

As used herein, “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, isotonic and absorption delaying agents,sweeteners and the like, suitable for administration to an individual.These pharmaceutically acceptable carriers may be prepared from a widerange of materials including, but not limited to, diluents, binders andadhesives, lubricants, disintegrants, coloring agents, bulking agents,flavoring agents, sweetening agents and miscellaneous materials such asbuffers and absorbents that may be needed in order to prepare aparticular therapeutic composition. The use of such media and agents forpharmaceutically active substances is well known in the art. It isunderstood that formulations contain components that are compatible withthe active ingredients. In one embodiment, talc, and magnesium stearateare included in the formulation. Other ingredients known to affect themanufacture of a composition of the invention as a dietary bar orfunctional food can include flavorings, sugars, amino-sugars, proteinsand/or modified starches, as well as fats and oils.

Dietary supplements, lotions or therapeutic compositions of embodimentsof the invention can be formulated in any manner known by one of skillin the art. In one embodiment, the composition is formulated into acapsule or tablet using techniques available to one of skill in the art.In capsule or tablet form, the recommended daily dose for an adult humanor animal can be contained in one to six capsules or tablets. Thecompositions can also be formulated in other convenient forms, such asan injectable solution or suspension, a spray solution or suspension, alotion, gum, lozenge, food or snack item. Food, snack, gum or lozengeitems can include any ingestible ingredient, including sweeteners,flavorings, oils, starches, proteins, fruits or fruit extracts,vegetables or vegetable extracts, grains, animal fats or proteins. Thus,compositions of the invention can be formulated into cereals, snackitems such as chips, bars, gumdrops, chewable candies or slowlydissolving lozenges. Compositions of the invention can be used for thetreatment of inflammation-based diseases, both acute and chronic.Particularly useful formulations of compositions of the invention canreduce the inflammatory response and thereby promote healing of, orprevent further damage to, the affected tissue. A pharmaceuticallyacceptable carrier can also be used in the compositions and formulationsof the invention.

The invention also provides a method of treatment of patients sufferingfrom, or susceptible to, ulcerogenic type disorders of the stomach andintestines, particularly acute and chronic gastric and duodenal ulcersand related conditions, which comprise administering to the patient aneffective amount of hops derivatives optionally together with additionalactive ingredients such as analgesic and/or anti-inflammatory compoundsor drugs.

Compositions of the invention can be used, for example, for thetreatment of inflammation in a subject, and for treatment of otherinflammation-associated disorders, such as an analgesic in the treatmentof pain and headaches, or as an antipyretic for the treatment of fever.Compositions of the invention can be used to treat arthritis, includingbut not limited to rheumatoid arthritis, spondyloathopathies, goutyarthritis, osteoarthritis, systemic lupus erythematosis, and juvenilearthritis.

Compositions of the invention can be used to prevent or treatNSAID-induced gastropathy in a mammal. For example, chronic, oral dosingof aspirin is associated with the development of ulcers. Whenadministered with the present invention, ulcer development can beameliorated or averted.

As disclosed herein, NSAID inhibition of gastric PGE₂ biosynthesis issignificantly attenuated with concomitant exposure to hops derivatives(see Examples 5 and 6). As further disclosed herein, the combination ofhops derivatives and NSAIDS exhibit increased therapeutic indices (seeExamples 7 and 8). Hops derivatives were also found to decrease ulcerformation and inhibit NSAID-induced gastric damage (see examples 9 and10). Therefore, the formation of gastric ulceration can be ameliorated,prevented or halted without negatively affecting the anti-inflammatoryactivity of the NSAID. Since compositions of the invention can affectNSAID gastropathy, embodiments can also be useful for treating andpreventing a variety of disorders including, but not limited to,autoimmune, inflammatory, neurological, infectious and cardiovasculardiseases, and cancers.

The invention provides a composition containing a fraction isolated orderived from hops, as disclosed herein, in combination with a secondcomponent. In one embodiment, the invention provides a compositioncomprising a fraction isolated or derived from hops and a non-aspirin,non-steroidal anti-inflammatory compound. The fraction isolated orderived from hops can be selected from the group consisting of alphaacids, isoalpha acids, reduced isoalpha acids, tetra-hydroisoalphaacids, hexa-hydroisoalpha acids, beta acids, and spent hops.

The fraction isolated or derived from hops can also be a compound of asupragenus having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and p orbital, with the proviso that if one of R, T, X, orZ is a p orbital, then the adjacent R, T, X, or Z is also a p orbital,thereby forming a double bond.

In still another embodiment, the fraction isolated or derived from hopscan contain a compound of Genus A having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In yet another embodiment of the ivnention, the fraction isolated orderived from hops can be a compound of Genus B having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

In an additional embodiment, the fraction isolated or derived from hopscan be a compound selected from the group consisting of humulone,cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. A composition of the invention can containspecific ranges of the active components, as disclosed herein.

A composition of the invention that contains a fraction isolated orderived from hops can be combined with a non-aspirin, nonsteroidalanti-inflammatory compound. Such a compound can be selected from thegroup consisting of salicylic acid, methyl salicylate, difulunisal,salsalate, olsalazine, sulfasalazine, acetanilide, acetaminophen,phenacetin, mefenamic acid, sodium meclofenamate, tolmetin, ketorolac,diclofenac, ibuprofen, naproxen, sodium daproxen, fenoprofen,ketoprofen, flurbioprofen, oxaprozin, piroxicam, meloxicam, tenoxicam,ampiroxicam, droxicam, pivoxicam, phenylbutazone, oxyphenbutazone,anitpyrine, aminopyrine, dipyrone, celecoxib, rofecoxib, nabumetone,apazone, nimensulide, indomethacin, sulindac, and etodolac. Acomposition of the invention can further comprises a pharmaceuticallyacceptable carrier. Such a composition can be formulated foradministration orally, topically, parenterally, or rectally.

In another embodiment, the invention provides a composition comprising areduced isoalpha acid isolated from hops and a non-steroidalanti-inflammatory compound. The reduced isoalpha acid can be, forexample, dihydro-isohumulone, dihydro-isocohumulone, anddihydro-adhumulone.

The invention additionally provides methods using the compositions ofthe invention disclosed herein. In one embodiment, the inventionprovides a method of producing an analgesic and an anti-ulcerogeniceffect in a mammal, comprising administering to the mammal an amount ofa fraction isolated or derived from hops sufficient to produce ananalgesic and anti-ulcerogenic effect and a nonsteroidalanti-inflammatory compound, whereby administration of the fractionisolated or derived from hops reduces gastric toxicity associated withthe non-steroidal anti-inflammatory compound. As disclosed herein, thefraction isolated or derived from hops can be administered concomitantlywith the non-steroidal anti-inflammatory compound. Alternatively, thefraction isolated or derived from hops can be administered after theadministration of the non-steroidal anti-inflammatory compound or beforethe administration of the non-steroidal anti-inflammatory compound.

In another embodiment, the invention provides a method of reducinggastric toxicity associated with a non-steroidal anti-inflammatorycompound by administering a fraction isolated or derived from hops to anindividual being treated with a non-steroidal anti-inflammatorycompound. The invention additionally provides a method of reducinggastroenteropathy by administering a fraction isolated or derived fromhops to an individual exhibiting a sign or symptom associated withgastroenteropathy The gastroenteropathy can, for example, involveulceration. The ulceration can be induced, for example, by food, anherb, bacteria, fungi or a drug. It is understood that a method of theinvention can be used to ameliorate a sign or symptom associated withgastropathy, gastroenteropathy, or gastric distress or discomfortassociated with a gastrointestintal irritant, as disclosed herein.

In one embodiment, the invention provides a method of reducing gastrictoxicity associated with a non-steroidal anti-inflammatory compound suchas a NSAID by administering a fraction isolated or derived from hops toan individual being treated with a non-steroidal anti-inflammatorycompound. A method can similarly be used to reduce gastric toxicity orgastroenterpathy associated with a gastrointestinal irritant. Varioushops derivatives or a fraction isolated or derived from hops can be usedto reduce gastric toxicity associated with a non-steroidalanti-inflammatory compound. The fraction isolated or derived from hops,for example, isoalpha acid or reduced isoalpha acid, can be administeredconcomitantly with a non-steroidal anti-inflammatory compound such as aNSAID. Such a concomitant administration can be in the same formulationor in different forumlations. Alternatively, the hops derivatives orfraction isolated or derived from hops can be administered after anon-steroidal anti-inflammatory compound has been administered, forexample, within a few minutes to a few hours to a few days ofadministration of a non-steroidal anti-inflammatory compound. Thefraction isolated or derived from hops can also be administered aftergastropathy from taking a non-steroidal anti-inflammatory compound hasdeveloped. In addition, a hops derivative or fraction isolated orderived from hops can be administered before a non-steroidalanti-inflammatory compound is administered, for example, as aprophylactic to prevent or reduce the severity of onset of non-steroidalanti-inflammatory compound-induced gastropathy. Such administration canbe a few minutes to a few hours to a few days prior to administration ofa non-steroidal anti-inflammatory compound. If the fraction isolated orderived from hops is administered prior to administration of anon-steroidal anti-inflammatory compound, it is understood that theadministration is within a timeframe such that the administration of thefraction isolated or derived from hops is effective at amelioratingnon-steroidal anti-inflammatory compound-induced gastropathy or reducingthe severity of the onset of non-steroidal anti-inflammatorycompound-induced gastropathy. The fraction isolated or derived from hopsis generally administered less than 7 days prior to the initialadministration of the non-steroidal anti-inflammatory compound, forexample, less than 6 days, less than 5 days, less than 4 days, less than3 days, or less than 2 days prior to administration of the non-steroidalanti-inflammatory compound. In particular, the fraction isolated orderived from hops can be administered within 24 hours of administrationof the non-steroidal anti-inflammatory compound.

Besides being useful for human treatment, embodiments of the inventionare also useful for treatment of other animals, including horses, dogs,cats, birds, sheep, pigs, and the like. Formulations for the treatmentof inflammation can inhibit the induction and activity of COX-2 withlittle effect on the synthesis of PGE₂ in the gastric mucosa.Historically, the NSAIDs used for treatment of inflammation lacked thespecificity of inhibiting COX-2 without affecting PGE₂ synthesis ingastric mucosal cells. Therefore, these drugs irritated and damaged thegastrointestinal system when used for extended periods. Suchcontraindications are not associated with the present invention andtherefore, the formulations described may be used for extended periodswith limited or no gastropathy. Administration can be by any methodavailable to the skilled artisan, for example, by oral, topical,transdermal, transmucosal, or parenteral routes.

As used herein, “reducing inflammation” refers to decreasing,ameliorating or inhibiting an inflammatory response. One skilled in theart can readily recognize a reduction in a sign or symptom associatedwith an inflammatory response. Reducing inflammation can refer todecreasing the severity of a sign or symptom associated withinflammation as well as inhibiting inflammation so that few or nosymptoms associated with inflammation are presented. As used herein,“gastropathy” refers to a disease of the stomach. As used herein,“gastroenteropathy” refers to a disorder of the alimentary canal, thetubular passage that extends from the mouth to the anus and functions indigestion and absorption of food and elimination of residual waste. Asused herein, “ulcerogenic disorder” refers to a disorder involving agastrointestinal ulcer. The gastrointestinal ulcer can, for example,result from exposure to an ulcerogenic substance such as an ulcerogenicchemical, an ulcerogenic environmental stimulus, an infection such as abacterial infection, for example, Helicobacter pylori, or astressed-induced ulcer. Such an ulcerogenic substance can be, forexample, a drug or compound such as a non-steroidal anti-inflammatorycompound or NSAID, food, an herb, bacteria, fungi, or other stimuli thatinduce ulcers. For example, the drug Fosmax is used to treatosteoporosis and causes adverse gastrointestinal effects. Therefore, acomposition of the invention can be used to ameliorate gastroenteropathycaused by a gastrointestinal irritant such as a drug. Although theinvention is exemplified with ameliorating gastropathy associated withNSAIDs, it is understood that the composition and methods disclosedherein can similarly be used for ameliorating gastropathy and/orgastroenteropathy associated with a variety of gastrointestinalirritants.

As disclosed herein, a composition of the invention containing afraction isolated or derived from hops, and optionally in combinationwith other components, can ameliorate a sign or symptom associated withgastropathy and/or gastroenteropathy. It is understood by those skilledin the art that a composition of the invention can be advantageouslyused to ameliorate gastroenteropathy associated with chemical,environmental, infection and/or emotionally stressful insults. Forexample, a composition of the invention can be used to ameliorategastropathy and/or gastroenteropathy associated with a gastrointestinalirritant. Such an irritant can include, for example, spices used forseasoning food, herbal preparations, alcohol, tobacco, stress, or otherwell known gastrointestinal irritants. A number of gastrointestinalirritants are well known to those skilled in the art, including, but notlimited to, foods and herbal preparations, including spices; stress andlifestyle, drugs, bacteria such as H. pylori and ulcer formation, andthe like, (see, for example, Myers et al., Am. J. Gastroenterol.82:211-214 (1987); Pippa et al., Scand. J. Gastroenterol. Suppl.167:32-35 (1989); Sivri, Fundam. Clinic. Pharmacol. 18:23-31 (2004);Bermejo et al., Rev. Esp. Enferm. Dig. 95: 621-624 and 625-628 (2003)).

As disclosed herein, various spices have been found to be inhibitory ofPGE₂ in the AGS cell model (see Examples 14-16). In combination withRIAA or other hops derivatives, the spices are expected to be lessgastrotoxic. For example, both ginger and capsasin analyzed in RAW andAGS cell models show inhibitory activity. Hence, RIAA, or otherfractions isolated or derived from hops, can antagonize activity innon-target cells (for example, gastric cells) and synergize in targetcells (for example inflammatory cells). Therefore, a composition of theinvention containing a fraction isolated or derived from hops can beused to decrease gastric toxicity and/or gastric discomfort associatedwith a gastric irritant such as a spice. For example, an IC₅₀ of 4 wasobserved for rosemary, an IC50>25 was observed for RIAA, and an IC50>25for the combination, which shows that RIAA can safen a spice. Thus, acomposition of the invention can be ingested with a spice, or mixed witha spice prior to ingestion or addition to food. In addition, acomposition of the invention can be administered in the form of chewinggum to alleviate gastric distress associated with ingestion of a spice.Such administration can similarly be applied to other gastric irritantsthat induce gastrotoxicity and/or gastropathy.

A composition of the invention containing a fraction isolated or derivedfrom hops, which have antimicrobial activities, can be used to botheradicate a gastric ulcer and promote gastric healing. In anotherembodiment of the invention, a composition of the invention can be usedin the form of an ointment or hypoallergenic paste or cream to treatulcerations or wounds of the skin or tissues, including dentalapplications in mouth ulcers.

As disclosed herein, a fraction isolated or derived from hops can beused to ameliorate a sign or symptom associated with gastroenteropathy,for examples, ulcers formed in the stomach or intestine. For example,the invention provides a method of reducing a sign or symptom associatedwith gastroenteropathy by administering a fraction isolated or derivedfrom hops to an individual exhibiting a sign or symptom associated withgastroenteropathy.

In addition to a fraction isolated or derived from hops, a compositionof the invention can further comprise a second component. An example ofsuch a second component is rosemary, an extract or compound derived fromrosemary. A composition of the invention can thus contain a fractionisolated or derived from hops and can further comprise an effectiveamount of rosemary, rosemary extract, or compounds derived fromrosemary. Thus, in addition to a fraction isolated or derived from hops,a composition of the invention can further contain rosemary, rosemaryextract, or those compounds known to be found in rosemary or extracts ofrosemary. These include 1,8-cineole, 19-alpha-hydroxyursolic acid,2-β-hydroxyoleanolic acid, 3-O-acetyloleanolic acid, 3-O-acetylursolicacid, 6-methoxy-luteolin-7-glucoside, 6-methoxyluteolin,6-methoxyluteolin-7-glucoside, methoxyluteolin-7-methylether,7-ethoxy-rosmanol, 7-methoxy-rosmanol, alpha-amyrin, alpha-humulene,alpha-hydroxyhydrocaffeic acid, alpha-pinene, alpha-terpinene,alpha-terpinenyl acetate, alpha-terpineol, alpha-thujone, apigenin,apigenin-7-glucoside, curcumene, benzyl-alcohol, β-amyrenone, β-amyrin,β-elemene, β-pinene, betulin, betulinic acid, bomeol, bomyl-acetate,caffeic acid, camphene, camphor, carnosic acid, camosol, carvacrol,carvone, caryophyllene, caryophyllene-oxide, chlorogenic acid,diosmetin, gamma-terpinene, hesperidin, isoborneol, limonene, luteolin,luteolin-3′-O-(3″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-(4″-O-acetyl)-β-D-glucuronide,luteolin-3′-O-β-D-glucuronide, luteolin-7-glucoside, methyl-eugenol,myrcene, neo-chlorogenic acid, nepetin, octanoic acid, oleanolic acid,p-cymene, piperitenone, rosmanol, rosmaric acid, rosmaricine,rosmaridiphenol, rosemarinic acid, rosmarinol, rosmariquinone, sabinene,sabinyl acetate, salicylates, salicylic acid-2-β-D-glucoside, squalene,terpinen-4-ol, terpinolene, thymol, trans-anethole, trans-carveol,ursolic acid, verbenone, and zingiberene.

In a composition containing a fraction isolated or derived from hops androsemary or an extract or compound derived therefrom, the compositioncan be formulated to deliver about 0.5 to 5000 mg of rosemary, anextract of rosemary, or rosemary-derived compound per day. Inparticular, an effective daily dose can be formulated to deliver about 5to 2000 mg of rosemary, an extract of rosemary, or rosemary-derivedcompound per day. For example, the composition can be formulated toprovide an effective daily dose to be administered once or twice a day.In a particular embodiment, a composition can contain about 75 mg ofrosemary extract or rosemary-derived compound or derivative, to beadministered once or twice a day.

A composition of the invention can further include a triterpene, such asoleanolic acid. In a particular embodiment, the composition can containabout 0.01 to 500 mg of rosemary extract and about 0.01 to 500 mg ofoleanolic acid. For example, a particular embodiment provides acomposition capable of producing concentrations in target tissues of 0.1to 10 μg/g tissue of rosemary extract and about 0.1 to 25 μg/g tissue ofoleanolic acid.

In still a further embodiment, a composition of the invention canfurther contain a triterpene species that is selected from the groupconsisting of 18-a-glycyrrhetinic acid, 18-β-glycyrrhetinic acid,2-a-3-a-dihydrooxyurs-12-3n-28-onic acid, 3-a-hydroxyursolic acid,3-oxo-ursolic acid, betulin, betulinic acid, celastrol, eburicoic acid,friedelin, glycyrrhizin, gypsogenin, oleanolic acid, oleanolicacid-3-acetate, pachymic acid, pinicolic acid, sophoradiol,soyasapogenol A, soyasapogenol B, tripterin, triptophenolide, tumulosicacid, ursolic acid, ursolic acid-3-acetate, uvaol, and β-sitosterol. Thetriterpene species can optionally be conjugated to a member selectedfrom the group consisting of mono- or di-saccharides, amino acids,sulfates, succinate, acetate, and glutathione.

In a particular embodiment, the composition can comprise about 0.5 to10000 mg or about 50 to 7500 mg of the fraction isolated or derived fromhops. Moreover, the composition can comprise, in addition to a fractionisolated or derived from hops, about 0.5 to 5000 mg of a secondcomponent, or about 5 to 2000 mg of a second component, wherein thesecond component is selected from the group consisting of rosemary,extract derived from rosemary, and a compound derived from rosemary. Inaddition, the composition can comprise about 0.001 to 10 weight percentof a first component containing a fraction isolated or derived fromhops, or about 0.1 to 1 weight percent of the first component. Also, thecomposition can comprise about 0.001 to 10 weight percent of a secondcomponent selected from rosemary, a rosemary extract or a compoundderived from rosemary, or about 0.1 to 1 weight percent of the secondcomponent. In another embodiment, a ratio of the first hops component tothe second rosemary component can be in the range of about 100:1 toabout 1:100, or in the range of about 50:1 to about 1:50

As disclosed herein (see Example 13), a composition containing afraction isolated or derived from hops does not increase fecalcalprotectin, in contrast to anti-inflammatory drugs such as NSAIDSwhich increase fecal calprotectin, an indicator of gastrointestinalinflammation. Such a composition containing a fraction isolated orderived from hops also contained rosemary extract and oleonolic acid inthe particular embodiment examined (Example 13). Therefore, acomposition containing a fraction isolated or derived from hops can beused to inhibit PGE₂ synthesis similar to NSAIDS while minimizinggastrointestinal inflammation.

Calprotectin is a calcium binding protein that has been found to haveantimicrobial, antifungal, and antiproliferative properties and topromote apoptosis in transformed and normal cells (Yui et al., Biol.Pharm. Bull. 26:753-760 (2003); Poullis et al., J. Gastroenterol.Hepatol. 18:756-762 (2003)). At least some of its activity appears toinvolve the ability of calprotectin to sequester zinc, causing alocalized zinc deficiency. However, data also indicate it haszinc-independent activities as well.

Calprotectin comprises 40% of the cytoplasmic protein in neutrophils,which infiltrate inflammatory sites, where they release the calprotectin(Yui et al., supra, 2003; Poullis et al., supra, 2003). It is also foundin blood monocytes and tissue macrophages at sites of acuteinflammation, whereas macrophages present in chronic inflammation andresident macrophages are negative for calprotectin (Yui et al., supra,2003). Calprotectin has also been found in mucosal squamous epithelialcells and cytokine-stimulated cultured keratinocytes, suggesting thatother cells may also produce this protein during inflammation (Schjervenet al., Br. J. Dermatol. 149:484-491 (2003)).

Calprotectin can be detected in plasma, urine, and several other bodilyfluids. In normal individuals, neutrophils migrate through the mucosalmembrane of the intestinal tract at the termination of their life and,therefore, a low level of fecal calprotectin is generally present.44.Tibble and Bjamason,. Drugs Today, 37:85-96 (2001). Since it isresistant to degradation, calprotectin can also be reproduciblyquantified in fecal samples (Røseth, A G. Digest. Liver Dis. 35:607-609(2003).

Interest in fecal calprotectin as a diagnostic test has grown since ithas been shown to be elevated in patients with gastrointestinalinflammation. For example, fecal calprotectin is significantly increasedin Inflammatory Bowel Disease (IBD; >100 μg/g stool), and a positiveassociation has also been observed between active IBD and increasedcalprotectin over quiescent disease (Costa et al., Digest. Liver Dis.35:642-647 (2003). Research suggests that fecal calprotectin is alsoincreased inflammation associated with colon cancer. Data comparinghealthy subjects with those having Irritable Bowel Syndrome(non-inflammatory disease) and IBD suggest a cutoff indicating thepresence of an organic disease is around 60 μg/g stool (Costa et al.,supra, 2003; Carroccio et al., Clin. Chem. 49:861-867 (2003)).

Non-steroidal anti-inflammatory drugs NSAIDs can induce gastrointestinaldamage, and long-term use causes inflammatory changes of thegastroduodenenal region in a high percentage of patients. Some studiessuggest that up to 65% of patients taking NSAIDs regularly for more than6 months will develop enteropathy (Tibble and Bjamason, supra, 2001).Damage to the gastrointestinal tract appears to occur rapidly with NSAIDuse. For example, in one study, 19% of subjects developed gastroduodenalulcers within 4 weeks of naproxen treatment (500 mg bid), and 41% ofpatients developed ulcers after 12 weeks of treatment (Goldstein et al.,Am. J. Gastroenterol. 96:1019-1027 (2001)).

Since fecal calprotectin is an indicator of gastrointestinalinflammation, several reports have investigated the use of fecalcalprotectin to detect damage from NSAIDs. In one study, fecalcalprotectin was reproducibly increased over 2-fold after 7 days ofnaproxen treatment (Meling et al., Scand. J. Gastroenterol. 31:339-344(1996)). These authors also reported that the increase in calprotectinwas positively correlated to gastroduodenal mucosal inflammation asassessed by endoscopy; however, a subsequent study with naproxenreproduced the increase in calprotectin but not the endoscopy findings(Shah et al., Gut 48:339-346 (2001)). Fecal calprotectin has been foundto be increased in 44% of subjects on chronic NSAID use, and thisincrease correlated significantly with 4-day excretion of ¹¹¹In-labelledwhite cells (Tibble et al., Gut 45:362-366 (1999)). Taken together,these studies suggest fecal calprotectin may be a sensitive, early-stagemarker of gastroduodenal damage from inflammation, such as seen withNSAIDs. Accordingly, a clinical trial was conducted to determine theeffect of a composition containing a fraction isolated or derived fromhops on calprotectin as a measure of gastrointestinal inflammation (seeExample 13).

Assay using AGS Cell Line

The discovery of COX-2 has made possible the design of drugs that reduceinflammation without removing the protective prostaglandins (PGs) in thestomach and kidney made by COX-1. As disclosed herein, compositions ofthe invention can be assessed using in vitro animal cells to assessCOX-2 and COX-1 inhibitory activity employing PGE₂, which hascytoprotective actions and plays a role in maintaining the integrity ofthe gastrointestinal mucosa, as an endpoint. Secondarily, different celltypes are used to confirm results. The screening process can be used toindicate compositions that have specific COX-2 activity and limitedCOX-1 inhibition. Compositions of embodiments of the invention can betested in two cell types: 1) human pulmonary cells or other cell line todetermine and identify optimal amounts and ratios for compositionscomprising more than one component; and 2) human gastric epithelialcells (AGS cell line), a gastrointestinal tract cell line and a modelsystem for assessing toxicity that is typically related to inhibition ofCOX-1, which is required for wound healing (such as ulcers). Hence,compositions of embodiments of the invention that can inhibit COX-2 orCOX-2 induction can be screened by selecting compositions that have lowor no activity in AGS cells and good activity in human pulmonary cellsor other cell lines.

As disclosed herein, a variety of assays are available to show theeffectiveness of one or more fractions isolated or derived from hops(see examples). It is understood by those skilled in the art that afraction isolated or derived from hops, as disclosed herein, can beassayed for activity in ameliorating gastric toxicity and/orgastroenteropathy using a variety of assays well known to those skilledin the art, including those exemplified herein.

The following examples are intended to illustrate but are not intendedto limit the scope of the invention.

EXAMPLE 1 AGS Gastric Mucosal Cells Constitutively Express bothCyclooxygenase-1 and Cyclooxygenase-2

Summary—This example demonstrates that the AGS human gastric mucosalcell line, possessing constitutive expression of COX-1 and COX-2, is amodel for assessing the gastrointestinal toxicity ofcyclooxygenase-inhibiting compounds.

Equipment used in this example included: an OHAS Model #E01140analytical balance, a Form a Model #F1214 biosafety cabinet (Marietta,Ohio), various pipettes to deliver 0.1 to 100 μL (VWR, Rochester, N.Y.),a cell hand tally counter (VWR Catalog #23609-102, Rochester, N.Y.), aForm a Model #F3210 CO₂ incubator (Marietta, Ohio), a hemacytometer(Hausser Model #1492, Horsham, Pa.), a Leica Model #DM IL invertedmicroscope (Wetzlar, Germany), a PURELAB Plus Water Polishing System(U.S. Filter, Lowell, Mass.), a 4° C. refrigerator (Form a Model #F3775,Marietta, Ohio), a vortex mixer (VWR Catalog #33994-306, Rochester,N.Y.), and a 37° C. water bath (Shel Lab Model #1203, Cornelius, Oreg.).

Chemicals and reagents—Prostaglandin E₂ EIA kit Monoclonal was purchasedfrom Cayman Chemical (Ann Arbor, Mich.). Anti-COX-1 and anti-COX-2rabbit polyclonal antisera were obtained from Upstate Biotechnology(CITY, NY); donkey anti-goat IgG-HRP was procured from Santa CruzBiotechnology (City, Calif.). Heat inactivated Fetal Bovine Serum(FBS-HI Cat. #35-011 CV), and Dulbeco's Modification of Eagle's Medium(DMEM Cat #10-013CV) was purchased from Mediatech (Hemdon, Va.). Allstandard reagents were obtained from Sigma (St. Louis, Mo.) and were thepurest commercially available.

Cell Culture—The human gastric mucosal cell line AGS was obtained fromthe American Type Culture Collection (ATCC number CRL-1739; Manassas,Va.) and sub-cultured according to the instructions of the supplier. Thecells were routinely cultured at 37° C. with 5% CO₂ in RPMI 1640containing 10% FBS, with 50 units penicillin/mL, 50 μg streptomycin/mL,5% sodium pyruvate, and 5% L-glutamine. Exponentially growing cells wereseeded into 6-well plates and grown to confluence. A 20 μL aliquot ofthe supernatant media was sampled for determination of PGE₂ content.Cells were then washed in PBS, scraped and lysed for immunoblotting.

Protein assay—Protein concentrations of cell lysates were determinedusing the NanoOrange Protein Quantitation Kit with bovine serum albuminas the standard (Molecular Probes, Eugene, OE) according to theprocedure supplied by the manufacturer. Fluorescence was determinedusing a Packard FluoroCount, Model BF 10000 fluorometer with theexcitation filter set at 485 nm and emission filter set at 570 nm usingPackard PlateReader version 3.0 software. The I-Smart program providedwith the Packard PlateReader was used to calculate the proteinconcentration.

Immunoblotting—Western blotting of COX-1 and COX-2 was performed usingPAGEr™ Gold Precast Gels (Bio Whittaker Molecular Applications(Rockland, Me.). AGS cell lysates containing approximately 60 μg proteinwere loaded with Laemmli Sample Buffer into the wells of the gel in atotal volume of 30 μL. The vertical minigel electrophoresis chamberswere made by Savant Instruments Inc. (Holbrook, N.Y.), model MV 120.Gels were run at 40 mA/plate (constant current) at room temperatureuntil the bromophenol blue stain reached the bottom of the gel, aboutone hour. Gels were then blotted on the polyvinyl fluoride transfermembranes (Pall Corporation, Ann Arbor, Mich.), overnight, at 500 mA and4° C. Precision Protein Standard molecular weight markers, unstained,broad range (BioRad, Hercules, Calif.) were used. The BioWest Extendedduration chemiluminescent substrate, a non-isotopic, horseradishperoxidase substrate kit for Western blot detection (BioImaging Systems,Upland, Calif.) was used for protein visualization. Images of westernblots were acquired using a UVP Epi Chemi II Darkroom (BioImagingSystems), analyzed and enhanced by LabWorks™ Image Acquisition andAnalysis Software (BioImaging Systems).

PGE₂ assay—A commercial, non-radioactive procedure for quantification ofPGE₂ was employed (Caymen Chemical, Ann Arbor, Mich.) and therecommended procedure of the manufacturer was used without modification.Briefly, 25 μL of the medium, along with a serial dilution of PGE₂standard samples, were mixed with appropriate amounts ofacetylcholinesterase-labeled tracer and PGE₂ antiserum, and incubated atroom temperature for 18 h. After the wells were emptied and rinsed withwash buffer, 200 μL of Ellman's reagent containing substrate foracetylcholinesterase were added. The reaction was carried out on a slowshaker at room temperature for 1 h and the absorbance at 415 nm wasdetermined. The PGE₂ concentration was represented as picograms per 10⁵cells.

Results—As seen in FIG. 4, the AGS cell line constitutively expressesboth COX-1 and COX-2, with COX-1 expression approximately 4-timesgreater than COX-2 expression. PGE₂ synthesis in AGS cells over 18 h was660 μg/10⁵ cells. Thus, this example demonstrates that the AGS humangastric mucosal cell line, possessing constitutive expression of COX-1and COX-2, can serve as a model for assessing the gastrointestinaltoxicity of cyclooxygenase-inhibiting compounds.

In the past, the classical COX-2 hypothesis has downplayed the role ofCOX-2 expression in the gastrointestinal mucosa. While in normal gastricmucosa COX-1 is the predominant COX isozyme, as demonstrated in thisexample and in the literature, there is increasing evidence thatdetectable amount of COX-2 mRNA and protein are both constitutivelyexpressed and inducible in specific locations of the gastric mucosa inboth animals and humans (Halter et al. Gut 49:443-453 (2001)). Recentstudies in rats have shown that whereas selective inhibition of COX-1 orCOX-2 is not ulcerogenic, combined inhibition of both COX-1 and COX-2induces severe lesions in the stomach and small intestine comparablewith the effects of NSAID such as indomethacin. This observationsuggests an important contribution of COX-2 to the maintenance ofgastrointestinal mucosal integrity.

EXAMPLE 2 Inhibition of PGE7 Synthesis in AGS Gastric Mucosal Cells andA549 Pulmonary Cells by Nonsteroidal Anti-Inflammatory Drugs

Summary—This example illustrates that inhibition of PGE₂ synthesis inAGS gastric cells and A549 pulmonary cells by NSAIDs correlates withtheir observed relative clinical gastrictoxicity.

Chemicals—Commercial formulations of rofecoxib tablets and celecoxibcapsules were used. PGE₂ EIA kits were obtained from Cayman Chemical(Ann Arbor, Mich.). Anti-COX-1 and anti-COX-2 rabbit polyclonal antiserawere obtained from Upstate Biotechnology (Waltham, Mass.), and donkeyanti-goat IgG-HRP was procured from Santa Cruz Biotechnology (SantaCruz, Calif.). Heat Inactivated Fetal Bovine Serum (FBS-HI Cat. #35-011CV) and Dulbecco's Modification of Eagle's Medium (DMEM Cat #10-013CV)was purchased from Mediatech (Herndon, Va.). IL-1β and all standardchemicals and non-steroidal anti-inflammatory drugs (NSAIDs), unlessnoted, were obtained from Sigma (St Louis, Mo.) and were of the highestpurity commercially available. All other chemicals were obtained fromsuppliers as described in EXAMPLE 1.

Cells—A549 (human pulmonary epithelial; ATCC number CCL-185) and AGScells (human gastric mucosa; ATCC number CRL-1739) were obtained fromthe American Type Culture Collection (Manassas, Va.) and sub-culturedaccording to the instructions of the supplier. The cells were routinelycultured at 37° C. with 5% CO₂ in RPMI 1640 containing 10% FBS, with 50units penicillin/mL, 50 μg streptomycin/mL, 5% sodium pyruvate, and 5%L-glutamine. On the day of the experiments, exponentially growing cellswere harvested and washed with serum-free RPMI 1640.

The log phase A549 and AGS cells were plated at 8×10⁴ cells per well in0.2 mL growth medium per well in a 96-well tissue culture plate. For thedetermination of PGE₂ inhibition by the test compounds in A549 cells,the procedure of Warner et al., also known as the WHMA-COX-2 protocol(Warner et al. Proc. Natl. Acad. Sci. U.S.A. 96, 7563-7568 (1999)) wasfollowed with no modifications. Briefly, 24 hours after plating of theA549 cells, interleukin-1β (10 ng/mL) was added to induce the expressionof COX-2. After 24 hr, the cells were washed with serum-free RPMI 1640and the test materials, dissolved in DMSO and serum-free RPMI, wereadded to the wells to achieve final concentrations of 25, 5.0, 0.5 and0.05 μg/mL. Each concentration was run in duplicate. DMSO was added tothe control wells in an equal volume to that contained in the testwells. Sixty minutes later, A23187 (50 μM) was added to the wells torelease arachidonic acid. Twenty-five μL of media were sampled from thewells 30 minutes later for PGE₂ determination.

Non-stimulated AGS cells were used in these studies. Twenty-four hoursafter plating in the 96-well microtiter plates, the cells were washedwith serum-free RPMI 1640 and the test materials, dissolved in DMSO andserum-free RPMI, were added to the wells to achieve final concentrationsof 25, 5.0, 0.5 and 0.05 μg/mL. Each concentration was run in duplicate.DMSO was added to the control wells in an equal volume to that containedin the test wells. Sixty minutes later, the calcium ionophore A23187 wasadded to the wells to achieve a final concentration of 50 μM.Twenty-five 1L of media were sampled from the wells 30 minutes after theaddition of A23187 for PGE₂ determination.

Cell viability—Cell viability was assessed by a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-basedcolorimetric assay (Sigma, St. Louis, Mo.). The MTT solution was addeddirectly to the wells after sampling for PGE₂ determination. Theabsorbance of each well was read at 580 nm using an ELISA plate reader.No toxicity was observed at the highest concentrations tested for any ofthe compounds.

PGE₂ Assay—A commercial, non-radioactive procedure for quantification ofPGE₂ was employed (Cayman Chemical, Ann Arbor, Mich.) for thedetermination of PGE₂ and the recommended procedure of the manufacturerwas used without modification. Briefly, 50 μL of the supernatant culturemedium, along with a serial dilution of PGE₂ standard samples, weremixed with appropriate amounts of acetylcholinesterase-labeled tracerand PGE₂ antiserum and incubated at room temperature for 18 h.Afterwards, the wells in the PGE₂-assay microtiter plate were emptiedand rinsed with wash buffer, 200 μL of Ellman's reagent containingsubstrate for acetylcholinesterase were then added. The reaction wasperformed on a slow shaker at room temperature for 1 h, after whichabsorbance at 415 nm was determined in a Bio-Tek Instrument ELISA platereader (Model #Elx800, Winooski, Vt.). The manufacturer's specificationsfor this assay include an intra-assay coefficient of variation of <10%,cross reactivity with PGD₂ and PGF_(2α) of less than 1%, and linearityover the range of 10-1000 pg/mL. The PGE₂ concentration was recorded aspg PGE₂ per 10⁵ cells.

Calculations—The median inhibitory concentration (IC₅₀) for PGE₂synthesis was calculated using CalcuSyn (BIOSOFT, Ferguson, Mo.). Thisstatistical package performs multiple drug dose-effect calculationsusing the median effect methods described by Chou and Talaly, Adv.Enzyme Regul. 22:27-55. (1984), hereby incorporated by reference.

Briefly, the analysis correlates the “Dose” and the “Effect” in thesimplest possible form: fa/fu=(C/C_(m))^(m), where C is theconcentration or dose of the compound and Cm is the median-effectivedose signifying the potency. Cm is determined from the x-intercept ofthe median-effect plot. The fraction affected by the concentration ofthe test material is fa and the fraction unaffected by the concentrationis fu (fu=1−fa). The exponent m is the parameter signifying thesigmoidicity or shape of the dose-effect curve. It is estimated by theslope of the median-effect plot.

The median-effect plot is a graph of x=log(C) vs y=log(fa/fu) and isbased on the logarithmic form of Chou's median-effect equation. Thegoodness of fit for the data to the median-effect equation isrepresented by the linear correlation coefficient r of the median-effectplot. Usually, the experimental data from enzyme or receptor systemshave an r>0.96, from tissue culture an r>0.90 and from animal systems anr>0.85. In the cell-based studies reported here, all linear correlationcoefficients were greater than 0.90. Experiments were repeated threetimes on three different dates. The percent inhibition at each dose wasaveraged over the three independent experiments and used to calculatethe median inhibitory concentrations reported. The therapeutic index(TI) for gastrointestinal safety was computed as the ratioAGS_((IC50))/A549_((IC50)). Spearman's rank correlation coefficientr_(s) was computed to quantify the degree of association between invitro ranking of TI by the AGS model and ranking of clinically assessedNSAID gastropathy. The probability of a type I error was set at thenominal five percent level.

Results—The highly specific COX-2 inhibitor diisofluorophosphate (DIFP)exhibited a median inhibitory concentration in A549 cells of 6.5 μg/mLand marginally inhibited PGE₂ synthesis in AGS cells at the highestconcentration tested of 25 μg/mL. Extrapolation of the dose-responsecurve provided an estimate of the AGS IC₅₀ for DIFP of 359 μg/mL (Table3). Of the three COX-2 selective agents, rofecoxib was the only compoundto demonstrate target cell selectivity greater than unity (AGS IC₅₀/A549IC₅₀>1). Celecoxib and nimensulide, both of which demonstrate high COX-2selectivity in enzyme assays, surprisingly exhibited greater PGE₂inhibition in the AGS gastric cells than in the A549 target cells.

Consistent with demonstrated clinical gastric toxicity, ibuprofen,aspirin and indomethacin all inhibited PGE₂ synthesis in the AGS cellline to a greater extent than in the target A549 cells. Salicylic acidand acetaminophen were relatively inactive in both cell models. FIG. 5compares and ranks the log TI computed for the NSAIDs. Values to theright of 0 indicate decreasing probability of gastrointestinal effects,while values to the left of 0 indicate increasing probability ofgastrointestinal effects. The AGS gastric cell model provided rankestimates of GI toxicity significantly associated with clinical rankingsof NSAID gastropathy, respectively r_(s)=0.933, p<0.01; 0.783, p<0.01;0.683, p=0.05. The ranking of NSAIDs from lowest to greatest potentialfor GI toxicity wasrofecoxib<acetaminophen<nimensulide<celecoxib<salicylicacid<ibuprofen<aspirin<naproxen<indomethacin.

These results validate the use of the AGS gastric mucosal cell line toevaluate potential gastrointestinal toxicity of anti-inflammatory agentscapable of inhibiting the synthesis of PGE₂. They also demonstratecellular specificity in the action of COX-inhibiting compounds. A ratioof 1 for IC₅₀ AGS/IC₅₀ A549 indicates IC₅₀'s that are the same for boththe AGS cell and A549 cells. If the ratio is higher than 1 for IC₅₀AGS/IC₅₀ A549, then the inhibition of PGE₂ is lower for the AGS cells. Alower inhibition of PGE₂ in AGS cells is favorable because AGS cell lineexpresses more COX-1, which maintains mucosal homeostasis. TABLE 3Median inhibitory concentrations for PGE₂ synthesis of select NSAID inthe A549 (target) and AGS (nontarget) cell models†. IC50 A549 IC50 AGSCompounds [μM] [μM] AGS/A549 DIFP†† 6.5 359 55 (1.5-28)  (125-1022)Rofecoxib 0.24 5.5 23 (0.15-0.45) (2.7-11)  Celecoxib 0.21 0.063 0.30(0.01-4.2)  (0.02-0.22) Nimensulide 0.32 0.12 0.38 (0.16-0.65)(0.0081-1.7)   Naproxen 28 0.83 0.03  (1.3-600) (0.24-2.8)  Ibuprofen 122.8 0.23 (6.8-19)  (1.3-5.8) Aspirin 18 2.9 0.16  (3.0-106) (1.4-5.6)Salicylic acid 4246 1065 0.25  (355-50971)   (94-12217) Acetaminophen238 535 2.3  (6.62-9589)  (179-1616) Indomethacin 8.1 0.0042 0.00052(2.6-26)  (0.00042-0.039) †Parenthetic values are 95% confidence intervals of the IC₅₀ estimate.††DIFP = diisofluorophosphate.

EXAMPLE 3 Inhibition of PGE2 Synthesis 1N Stimulated and NonstimulatedMurine Macrophages by HOPS (Humulus lupulus) Compounds and Derviatives

Summary—This example illustrates that hops fractions and derivativesinhibit COX-2 synthesis of PGE₂ preferentially over COX-1 synthesis ofPGE₂ in the RAW 264.7 murine macrophage model.

Chemicals and reagents—Bacterial lipopolysaccharide (LPS; B E. coli055:B5) was from Sigma (St. Louis, Mo.). Hops fractions (1) alpha hop(1% alpha acids; AA), (2) aromahop OE (10% beta acids and 2% isomerizedalpha acids, (3) isohop (isomerized alpha acids; IAA), (4) beta acidsolution (beta acids BA), (5) hexahop gold (hexahydro isomerized alphaacids; HHIAA), (6) redihop (reduced isomerized-alpha acids; RIAA), (7)tetrahop (tetrahydro-iso-alpha acids THIAA) and (8) spent hops wereobtained from Betatech Hops Products (Washington, D.C., U.S.A.). Thespent hops were extracted two times with equal volumes of absoluteethanol. The ethanol was removed by heating at 40° C. until a only thickbrown residue remained. This residue was dissolved in DMSO for testingin RAW 264.7 cells. Unless otherwise noted, all standard reagents wereobtained from Sigma (St. Louis, Mo.) and were the purest commerciallyavailable. All other chemicals and equipment were as described inEXAMPLES 1 and 2.

Cell culture—RAW 264.7 cells, obtained from American Type CultureCollection (Catalog #TIB-71, Manassas, Va.), were grown in Dulbecco'sModification of Eagle's Medium (DMEM, Mediatech, Herndon, Va.) andmaintained in log phase. The DMEM growth medium was made by adding 50 mLof heat inactivated FBS and 5 mL of penicillin/streptomycin to a 500 mLbottle of DMEM and storing at 4° C. The growth medium was warmed to 37°C. in water bath before use.

On day one of the experiment, the log phase RAW 264.7 cells were platedat 8×10⁴ cells per well in 0.2 mL growth medium per well in a 96-welltissue culture plate in the morning. At the end of the day one (6 to 8 hpost plating), 100 μL of growth medium from each well were removed andreplaced with 100 μL fresh medium.

A 1.0 mg/mL stock solution of LPS, used to induce the expression ofCOX-2 in the RAW 264.7 cells, was prepared by dissolving 1.0 mg of LPSin 1 mL DMSO. It was vortexed until dissolved and stored at 4° C. Beforeuse, it was melted at room temperature or in a 37° C. water bath.

On day two of the experiment, test materials were prepared as 1000×stock in DMSO. In 1.7 mL microfuge tubes, 1 mL DMEM without FBS wasadded for test concentrations of 0.05, 0.10, 0.5, and 1.0 μg/mL. Two μLof the 1000× DMSO stock of the test material was added to the 1 mL ofmedium without FBS. The tube contained the final concentration of thetest material concentrated 2-fold and was placed in an incubator for 10minutes to equilibrate to 37° C.

For COX-2 associated PGE₂ synthesis, 100 μL of medium were removed fromeach well of the cell plates prepared on day one and replaced with 100μL of equilibrated 2× final concentration of the test compounds. Cellswere then incubated for 90 minutes. Twenty μL of LPS were added to eachwell of cells to be stimulated to achieve a final concentration of 10 ngLPS/mL and the cells were incubated for 4 h. Following the LPSstimulation, the appearance of the cells was observed and viability wasdetermined as described in EXAMPLE 2. No toxicity was observed at thehighest concentrations tested for any of the compounds. Twenty-five μLof supernatant medium from each well was transferred to a cleanmicrofuge tube for the determination of PGE₂ released into the medium.PGE₂ was assayed and reported as previously described in EXAMPLE 1.

For COX-1 associated PGE₂ synthesis, 100 μL of medium were removed fromeach well of the cell plates prepared on day one and replaced with 100μL of equilibrated 2× final concentration of the test compounds. Cellswere then incubated for 90 minutes. Next, instead of LPS stimulation,the cells were incubated with 100 μM arachidonic acid for 15 minutes.Twenty-five μL of supernatant medium from each well was transferred to aclean microfuge tube for the determination of PGE₂ released into themedium. The appearance of the cells was observed and viability wasdetermined as described in EXAMPLE 2. No toxicity was observed at thehighest concentrations tested for any of the compounds. Twenty-five μLof supernatant medium from each well was transferred to a cleanmicrofuge tube for the determination of PGE₂ released into the medium.PGE₂ was determined and reported as previously described in EXAMPLE 1.The median inhibitory concentrations (IC₅₀) for PGE₂ synthesis from bothCOX-2 and COX-1 were calculated as described in EXAMPLE 2. TABLE 4 COX-2and COX-1 inhibition in RAW 264.7 cells by hop fractions and derviativesCOX-2 IC₅₀ COX-1 IC₅₀ COX-1 Test Material [μg/mL] [μg/mL] IC₅₀/COX-2IC₅₀ Genus A structures Isohop (IAA) 0.13 18 144 Redihop (RIAA) 0.34 2987 Genus B structures Tetrahop (THIAA) 0.20 4.0 21 Hexahop (HHIAA) 0.293.0 11 Alpha acids Alpha hop (AA) 0.21 6.2 30 Others Aromahop OE 1.6 4.12.6 Beta acids (BA) 0.54 29 54 Spent hops (EtOH) 0.88 21 24

As seen in Table 4, all hops fractions and derivatives selectivelyinhibited COX-2 over COX-1 in this target macrophage model. This was anovel and unexpected finding. The extent of COX-2 selectivity for thehops derivatives IAA and RIAA, respectively, 144- and 87-fold, wasunanticipated. In this RAW 264.7 cell model, Genus A compounds exhibiteda greater COX-2 selectivity than Genus B compounds, averaging 116-foldvs 16-fold, respectively, greater COX-2 inhibition. Alpha acid, betaacids and spent hops were also highly selective COX-2 inhibitors withCOX-1/COX-2 ratios, respectively, 30, 54 and 24. Such high COX-2selectivity combined with low median inhibitory concentrations, has notbeen previously reported for natural products from other sources.Aromahop was least COX-2 selective with a COX-1/COX-2 ratio of 2.6,similar to acetaminophen (EXAMPLE 2) a NSAID with demonstrated lowclinical gastrotoxicity.

EXAMPLE 4 Lack of Inhibition of PGE₂ Synthesis in Gastric Mucosal Cellsby HOPS (Humulus lupulus) Compounds and Derviatives

Summary—This example illustrates the lack of PGE₂ inhibition by hopsfractions and in the AGS human gastric mucosal cell line implying lowgastric irritancy potential of these compounds.

Chemicals and reagents were used as described in EXAMPLE 3. AGS cellswere grown and used for testing hops compounds and derivatives asdescribed in EXAMPLE 2. PGE₂ was determined and reported as previouslydescribed in EXAMPLE 1. The median inhibitory concentrations (IC₅₀) forPGE₂ synthesis from AGS cells were calculated as described in EXAMPLE 2.TABLE 5 Median inhibitory concentrations (IC₅₀) for PGE₂ synthesis ofhops derivatives in the AGS cell model† AGS IC₅₀ 95% Confidence IntervalCompound [μg/mL] [μg/mL] r Alpha Acids Alphahop 17  2.4-124 0.927 GenusA structures†† Iso-alpha acids Isohop (IAA) 16 4.9-54  0.970 Isorich 9.21.1-81  0.894 Reduced iso-alpha acids Redihop (RIAA) 21  3.1-145 0.936Genus B structures††† Tetra-iso-alpha acids Tetrahop (THIAA) 51  6.8-3740.949 Hexa-iso-alpha acids Hexahop (HHIAA) 34 25-47 0.998 OthersBetaStab 73  18-291 0.977 Tannin extract #4411 59  11-324 0.963 Aromahop43 21-85 0.992 #1115 (Spent hops) 35  8.5-141 0.970 Positive ConcurrentControl Aspirin 1.2 (0.37-4.1)  0.950 Historical Control Aspirin 0.52(0.26-1.0)  — Ibuprofen 0.57 (0.27-1.2)  — Rofecoxib 1.8 (0.90-3.7)  —Celcoxib 0.024 (0.0068-0.082)  —†IC₅₀ values are computed from the average of three independent assays;AGS cells were plated and allowed to reach 80% confluence. Cells werewashed and test material was added 60 minutes prior to treatment withA23187. Thirty minutes later, media was removed for PGE2 determination.

Median inhibitory concentrations for PGE₂ synthesis of hops derivativesin the AGS cell model are presented in Table 3. Genus B structures, ingeneral, were less inhibitory than Genus A structures and alpha acids.In the Genus B group, IC₅₀ values for THIAA and HHIAA were,respectively, 51 and 34 μg/mL. IC₅₀ values for IAA, Isorich and RIAAfrom the Genus A group were, respectively, 16, 9.2 and 21 μg/mL, onaverage 63% lower than IC₅₀ values from Genus B species. With relativelyhigh IC₅₀ values, hops derivatives BetaStab (73 μg/mL), Tannin extract#4411 (59 μg/mL), Aromahop (43 μg/mL) and #1115 spent hops (35 μg/mL)would rank as non-irritating to the gastric mucosa. Unexpectedly, allhops derivatives were substantially less inhibitory to AGS gastricmucosal cells than any NSAID including the newer, highly selective COX-2drugs rofecoxib and celecoxib.

EXAMPLE 5 Combinations of Genus a HOPS Derivatives and Ibuprofen orAspirin Exhibit Decreased Inhibition of PGE) Synthesis in AGS GastricMucosal Cells

Summary—This example illustrates an antagonistic effect of Genus A hopsspecies on the inhibition of gastric PGE₂ by the NSAIDs ibuprofen andaspirin. The implication of this effect is that Genus A hops derivativesfunction to attenuate the gastropathy of NSAIDs.

Methods—Chemicals and reagents were used as described in EXAMPLES 2 and3. AGS cells were grown and used for testing the Genus A hops derivativeRIAA and combinations of RIAA:ibuprofen and RIAA:aspirin as described inEXAMPLE 2. RIAA:NSAID combinations were formulated to contain either 1,9, 50, 91, or 99 percent RIAA. Concentrations of test material of 50, 5,0.5 and 0.05 μg/mL were assayed in duplicate. PGE₂ was determined andreported as previously described in EXAMPLE 1. The median inhibitoryconcentrations (IC₅₀) for PGE₂ synthesis from AGS cells were calculatedas described in EXAMPLE 2.

Synergy or antagonism of test formulations was quantified using thecombination index (CI) parameter and CalcuSyn (BIOSOFT, Ferguson, Mo.)software. The CI of Chou-Talaly is based on the multiple drug-effect andis derived from enzyme kinetic models (Chou and Talalay, Adv. EnzymeRegul. 22:27-55 (1984)). The equation determines only the additiveeffect rather than synergism or antagonism. However, synergism, as usedherein, is defined as a more than expected additive effect, andantagonism as a less than expected additive effect as proposed by Choand Talalay Using the designation of CI=1 as the additive effect, formutually exclusive compounds that have the same mode of action or formutually non-exclusive drugs that have totally independent modes ofaction, the following relationships were obtained: CI<1, =1, and >1indicating synergism, additivity and antagonism, respectively.

Results—FIG. 6A and FIG. 6B depict the percent PGE₂ inhibition in theAGS gastric mucosal cell, respectively, for ibuprofen, RIAA andcombinations of RIAA:ibuprofen (FIG. 6A), and aspirin, RIAA andcombinations of RIAA:aspirin (FIG. 6B). Unexpectedly, as little as onepercent RIAA in combination with ibuprofen or aspirin reduces the PGE₂inhibitory effect of either NSAID. Computation of the CI for RIAA andibuprofen or aspirin indicates extremely strong synergy over the entiredose-response curve for RIAA:NSAID combinations of 100:1 to 1:10 (Table6). With RIAA:NSAID combinations of 100:1, inhibition of PGE₂biosynthesis was insufficient to compute a dose-response. TABLE 6Combination index† values for RIAA:ibuprofen and RIAA:aspirincombinations in the AGS gastric mucosal cell model Test Material CI IC₅₀CI IC₇₅ CI IC₉₀ Average CI Ibuprofen Combinations RIAA:ibuprofen (1:100)170 16 1.5 63 RIAA:ibuprofen (1:10) 164 12 1.1 59 RIAA:ibuprofen (1:1)210 60 43 104 RIAA:ibuprofen (10:1) 28 5.9 3.4 12 RIAA:ibuprofen (100:1)NDR†† Aspirin Combinations RIAA:aspirin (1:100) 24164 54835 4.0 × 10⁵1.3 × 10⁵ RIAA:aspirin (1:10) 254 2.7 0.5 85 RIAA:aspirin (1:1) 697 159776808 26367 RIAA:aspirin (10:1) 455 128 170 251 RIAA:aspirin (100:1)NDR †CI <1, =1, and >1 indicate synergism, additivity and antagonism,respectively.††NDR = no dose-response to 50 μg test material/mL over doses of 50, 5,0.5 and 0.05 μg test material/mL.

Similar experiments are performed with various fractions isolated orderived from hops and various non-steroidal anti-inflammatory compoundssuch as NSAIDs.

EXAMPLE 6 Combinations of Genus B HOPS Derivatives and Ibuprofen orAspirin Exhibit Decreased Inhibition of PGE2 Synthesis in AGS GastricMucosal Cells

Summary—This example illustrates an antagonistic effect of Genus B hopsspecies on the inhibition of gastric PGE₂ by the NSAIDs ibuprofen andaspirin. The implication of this effect is that Genus B hops derivativesfunction to attenuate the gastropathy of NSAIDs.

Methods—Chemicals, reagents and methods were as described in EXAMPLES 2,3 AND 5. AGS cells were grown and used for testing the Genus B hopsderivative THIAA and combinations of THIAA:ibuprofen and THIAA:aspirinas described in EXAMPLE 2. THIAA:NSAID combinations were formulated tocontain either 1, 9, 50, 91, or 99 percent THIAA. Concentrations of testmaterial of 50, 5, 0.5 and 0.05 μg/mL were assayed in duplicate. PGE₂was determined and reported as previously described in EXAMPLE 1. Themedian inhibitory concentrations (IC₅₀) for PGE₂ synthesis from AGScells were calculated as described in EXAMPLE 2. Synergy or antagonismof test formulations was quantified using the combination index (CI)parameter as described in EXAMPLE 5.

Results—FIGS. 7A and 7B depict the percent PGE₂ inhibition in the AGSgastric mucosal cell, respectively, for ibuprofen, THIAA andcombinations of THIAA:ibuprofen (FIG. 7A), and aspirin, RIAA andcombinations of RIAA:aspirin (FIG. 7B). THIAA combinations withibuprofen were qualitatively similar to RIAA combinations with ibuprofenwith respect to the attenuation of PGE₂ inhibition by the NSAID. CI forTHIAA:ibuprofen combinations, however, indicated antagonism only at theportions of the dose-response curve below 50 percent inhibition.Graphically as seen in FIG. 7B, the THIAA:aspirin combinations appear toincrease PGE₂ inhibition in AGS gastric mucosal cells at both the 5 and0.5 test material concentrations. Computation of CI revealed thatTHIAA:aspirin combinations were antagonistic at the low end of thedose-response curve where inhibition of PGE₂ was less than 40 percent.Above the 40 percent level, strong synergy was seen with THIAA andaspirin combinations. TABLE 7 Combination index† values forTHIAA:ibuprofen and THIAA:aspirin combinations in the AGS gastricmucosal cell model Test Material CI IC₅₀ CI IC₇₅ CI IC₉₀ Average CIIbuprofen Combinations THIAA:ibuprofen (1:100)^(a) 2.5 0.30 0.037 1.1THIAA:ibuprofen (1:10)^(b) 18 0.86 0.040 6.3 THIAA:ibuprofen (1:1)^(c)4.0 0.48 0.058 1.5 THIAA:ibuprofen (10:1)^(d) 1.1 0.083 0.0065 0.40THIAA:ibuprofen (100:1)^(d) 1.3 0.067 0.0038 0.46 Aspirin CombinationsTHIAA:aspirin (1:100)^(e) 0.27 0.0033 4.1 × 10⁻⁵ 1 × 10⁻⁵ THIAA:aspirin(1:10)^(f) 0.16 0.0018 2.5 × 10⁻⁵ 1 × 10⁻⁵ THIAA:aspirin (1:1)^(a) 7.960.08 0.0020 THIAA:aspirin (10:1)^(e) 0.24 0.0078 0.0011 THIAA:aspirin(100:1)^(f) 0.20 0.0086 0.00065†CI <1, =1, and >1 indicate synergism, additivity and antagonism,respectively.††NDR = no dose-response to 50 μg test material/mL over doses of 50, 5,0.5 and 0.05 μg test material/mL.^(a)Antagonism to IC₆₀;^(b)Antagonism to IC₇₀;^(c)Antagonism to IC₆₅;^(d)Antagonism to IC₅₀.^(e)Antagonism to IC₄₀;^(f)Antagonism to IC₃₅.

Similar experiments are performed with various fractions isolated orderived from hops and various non-steroidal anti-inflammatory compoundssuch as NSAIDs.

EXAMPLE 7 Combinations of Genus A HOPS Derivatives and Ibuprofen orAspirin Exhibit Increased Therapeutic Indicies

Summary—This example illustrates that combinations of Genus Arepresentative species RIAA increases the therapeutic index of bothibuprofen and aspirin.

Methods—Chemicals and reagents were used as described in EXAMPLES 2 and3. RAW 264.7 and AGS cells were grown and used for testing the Genus Ahops derivative RIAA and combinations of RIAA:ibuprofen and RIAA:aspirinas described in EXAMPLE 2 and 3. RIAA:NSAID combinations were formulatedto contain either 50 percent RIAA. Concentrations of test material of50, 5, 0.5 and 0.05 μg/mL were assayed in duplicate. PGE₂ was determinedand reported as previously described in EXAMPLE 1. Plots of log IC₅₀ratios (AGS/COX-2) were made as described in EXAMPLE 2.

Results—As seen in FIG. 8, with increasing RIAA in the test formulation,the potential for inducing gastrotoxicity was decreased for bothibuprofen FIG. 8A and aspirin FIG. 8B.

Similar experiments are performed with various fractions isolated orderived from hops and various non-steroidal anti-inflammatory compoundssuch as NSAIDs.

EXAMPLE 8 Combinations of Genus A HOPS Derivatives and Ibuprofen orAspirin Exhibit Increased Therapeutic Indicies

Summary—This example illustrates that combinations of Genus Brepresentative species THIAA increases the therapeutic index of bothibuprofen and aspirin. Thus, although THIAA species of hops derivativeswere antagonistic only at portions of the dose-response curve below 40percent, the overall effect was to increase the efficacy of NSAIDs intarget cells to a greater extent resulting in an unexpectedly large,positive increase in the therapeutic index of NSAIDs.

Methods—Chemicals and reagents were used as described in EXAMPLES 2 and3. RAW 264.7 and AGS cells were grown and used for testing the Genus Bhops derivative THIAA and combinations of THIAA:ibuprofen andTHIAA:aspirin as described in EXAMPLE 2 and 3. THIAA:NSAID combinationswere formulated to contain either 1 or 50 percent THIAA. Concentrationsof test material of 50, 5, 0.5 and 0.05 μg/mL were assayed in duplicate.PGE₂ was determined and reported as previously described in EXAMPLE 1.Plots of log IC₅₀ ratios (AGS/COX-2) were made as described in EXAMPLE2.

Results—As seen in FIG. 9, with increasing THIAA in the testformulation, the potential for inducing gastrotoxicity was decreased forboth ibuprofen FIG. 9A and aspirin FIG. 9B.

Similar experiments are performed with various fractions isolated orderived from hops and various non-steroidal anti-inflammatory compoundssuch as NSAIDs.

EXAMPLE 9 Activity of Genus A and Genus B HOPS Derivates AgainstStress—Induced Ulcers

Stress-induced ulcers are experimentally induced in rats by encasing theanimals in plaster of Paris bandages for 24 hours. The effect ofcompounds on formation of stress-induced ulcers is assessed by dosingthe test compound one hour before encasing the rats in the bandage andagain six hours after encasing the rats. After 24 hours, the bandage isremoved and the gastric damage is assessed compared to untreated rats.Hops derivatives used were as previously described in EXAMPLE 3.

Treating the rats with 100 mg of test material/kg results in inhibitionof the mean total ulcer score.

EXAMPLE 10 Influence of Genus A and Genus B HOPS Derivates on GastricDamage Caused by Administration of Nonsteroidal Anti-Inflammatory Drugsin Rats

Gastric damage caused by nonsteroidal, anti-inflammatory drugs isassessed by administering a dose of the test material followed later bya relatively high dose of the NSAID. After a further five hours, theextent of gastric damage is determined and percent inhibition iscalculated. Hops derivatives used were as previously described inEXAMPLE 3.

Substantial inhibition of NSAID-induced gastric damage is observed withall hops derivatives.

EXAMPLE 11 Acute Toxicity of Genus A and Genus B HOPS Derivates in Rats

The acute toxicity of hops derivatives is examined in rats. Ten, young,Fisher 344 male rats averaging 100 g are orally dosed with 5000 mg testmaterial/kg body weight and observed for 14 days; the number of deadrats is determined. The low acute toxicity of hops derivatives isillustrated by lack of lethality when administered to rats orally at5,000 mg test material/kg body weight.

EXAMPLE 12 Reduction of Acute Toxicity of Aspirin when Administered inConjunction with HOPS Derivatives

The ability of hops derivatives to reduce acute toxicity of aspirin isexamined in mice. Ten, young, male and female mice per group averaging12 g are orally dosed with 50, 100, 500, 1000, or 5000 mg testmaterial/kg body weight and observed for 14 days; the median lethal doseis computed as described in EXAMPLE 2. Oral administration of aspirin ora combination of aspirin and hops derivatives in a 1:1 ratio to male andfemale mice indicates a strong protective effect of the hopsderivatives. Hops combinations with aspirin decrease or preventlethality at all doses of aspirin.

Thus, among the various formulations taught there has been disclosed aformulation containing, as a first active component, a hops derivativedescribed by the supragenus disclosed and as a second component a NSAID.It will be readily apparent to those skilled in the art that variouschanges and modifications of an obvious nature may be made withoutdeparting from the spirit of the invention. Such changes andmodifications would include, but not be limited to, the incipientingredients added to affect the capsule, tablet, powder, lotion, food orbar manufacturing process as well as vitamins, flavorings and carriers.Other such changes or modifications would include the use of herbs orother botanical products containing the combinations of the preferredembodiments disclosed herein. Many additional modifications andvariations of the embodiments described herein may be made withoutdeparting from the scope, as is apparent to those skilled in the art.The specific embodiments described herein are offered by way of exampleonly.

In conclusion, one embodiment is a composition for treating orpreventing NSAID-induced gastropathy. The composition contains at leastone hops derivative and a NSAID. The administration may be enteral orparenteral.

EXAMPLE 13 Comparison of the Effects of a Composition Containing HOPSExtract Versus Anti-Inflammatory Medication

This example describes a randomized cross-over study to assess theeffect of a composition containing hopes extract and a non-steroidalanti-inflammatory (NSAID), naproxen, on fecal calprotectin.

Trial Design—The trial was a randomized, cross-over study to compare theeffects of IP2003-001CT and naproxen on fecal calprotectin. Subjectswere screened for participation at Visit 0 (V0). If selected forparticipation in the trial, subjects returned for the first visit (V1).Two background fecal calprotectin samples were obtained between V0 andV1. Subjects were randomized and placed on either Arm 1 or Arm 2 of thetrial. All subjects were instructed to abstain from alcohol ingestionfrom 1 week before trial initiation to the end of the entire trial.

Arm 1 subjects were assigned IP2003-001CT at 2 tablets bid (twice daily)at V1, which was taken for 14 days total. Visit 2 (V2) occurred at 1days after V1 and Visit 3 (V3) occurred at day 14 days after V1. After a21 day washout, subjects returned for Visit 4 (V4) and were assignednaproxen 500 mg bid for 14 days. Subjects returned for Visit 5 after 7days (V5; 42 days total) and the final visit, Visit 6 (V6) at 49 daystotal.

Arm 2 subjects were assigned naproxen 500 mg bid at V1, which was takenfor 14 days total. Visit 2 (V2) occurred at 7 days after V1, and Visit 3(V3) occurred 14 days after V1. After a 21 day washout, subjectsreturned for Visit 4 (V4) and were assigned IP2003-001CT at 2 tabletsbid for 14 days. Subjects returned for Visit 5 after 7 days (V5; 42 daystotal) and the final visit, Visit 6 (V6) at 49 days total.

Randomization was performed using a computer-generated randomizationtable (Microsoft Excel). Subjects were assigned to each arm on trialentry (V1).

Subjects were recruited through newspaper and radio advertisements. Menbetween ages 18 and 45 were initially screened by phone or e-mail.Inclusion criteria included body mass index (BMI) between 20 and 29kg/m² and no indication of recent or past gastrointestinal disease andhealthy as determined by normal standard blood tests.

Subjects were excluded if screening laboratory showed abnormal completeblood count (CBC), kidney or liver function markers, or blood glucosevalues. Subjects currently on prescriptive medications, and subjects whohad used NSAIDs or aspirin in the 2 weeks preceding or oralcorticosteroids in 4 weeks preceding trial initiation were excluded.Exclusion criteria also included allergy to any of the ingredients inthe supplement, naproxen, NSAIDs, or aspirin; a history of peptic ulcerdisease, gastritis, esophagitis, or liver, kidney, or heart disease;history of bleeding disorders; uncontrolled hypertension (blood pressure(BP)>140/90); diabetes; HIV; history of or active cancer (except skincancer); history of untreated endocrine, neurological, or infectiousdisorder; history of serious mental illness or episode of attemptedsuicide within preceding 10 years.

Investigational Product—Each tablet of IP2003-001 contained: 200 mgreduced iso-alpha acids (as magnesium salt from Humulus lupulus coneextract), 200 mg rosemary extract (Rosmarinus officinalis), and 40 mgoleanolic acid (from olive leaf extract Olea europaea) with excipientsof microcrystalline cellulose, silica, stearic acid, magnesium stearate,calcium silicate. Two of the ingredients in the tablets, rosemary andoleanolic acid, are included in the generally regarded as safe (GRAS)list by the United States Food and Drug Administration (FDA). Reducediso-alpha acids from hops (RIAA) are food grade substances that are usedas natural, foam-stabilizing and bittering compounds in beer making andhave a history of safe use.

Naproxen (2-naphthaleneacetic acid, 5 methoxy-α-methyl-(+)) is a NSAIDwith analgesic and antipyretic properties. Naproxen is rapidly andcompletely absorbed from the gastrointestinal tract; peak plasma levelsof naproxen are attained in 2 to 4 hours after administration, withsteady-state conditions normally achieved after 4 to 5 doses. Inclinical studies in arthritis patients, naproxen has been shown to becomparable to aspirin and indomethacin in controlling pain and jointstiffness. Adverse reactions of 3% or more occurrence with naproxeninclude: constipation, heartburn, abdominal pain, nausea, dyspepsia,diarrhea, stomatitis, headache, dizziness, drowsiness, lightheadedness,vertigo, itching (pruritus), skin eruption, ecchymoses, sweating,purpura, tinnitus, hearing disturbances, visual disturbances, edema,dyspnea, palpitations, and thirst.

Clinical and Laboratory Analysis—Blood was drawn at V0 for screeninglaboratories, and at V1, V3, V4, and V6 for high sensitivity C-reactiveprotein (hsCRP). Follow-up CBC and comprehensive metabolicpanel/complete metabolic profile (CMP) were performed at V3 and V6 formonitoring of subject baseline laboratories. General laboratory,including CBC and chemistry panels during screening and hsCRP, wereperformed by Laboratories Northwest (Tacoma, Wash.).

Fecal calprotectin assays were performed using the PhiCal enzymeimmunoassay (Great Smokies Diagnostic Laboratory, Asheville, N.C.). Thetest sensitivity is 15 μg/g stool (equivalent to 6.25 ng/mL) and thetest is linear to 250 μg/g stool (equivalent to 100.00 ng/mL). Withinrun variation is 6% coefficient of variation (CV), and day-to-dayvariation is 14% CV.

Subjects were provided kits to obtain stool samples at home and bringthem to each visit. Collection of stool samples was performed asdescribed in literature provided by the manufacturer (Great SmokiesDiagnostic Laboratory). Two stool samples were collected from thesubjects at V1 for intra-individual variation and baselinedetermination. One stool sample was collected at V2, V3, V4, V5, and V6.

First morning urine was collected at home by each subject before V1, V2,V3, V4, V5, and V6. Urine was frozen for assessment of inflammatorymediators.

Vital signs and general physical exam was performed at each visit. AtV2, V3, V4, V5, and V6, a general Tolerance Questionnaire was alsoobtained to assess for tolerance to the IP2003-001 and naproxen.

Statistical Methods—Data were analyzed by a one-way analysis of variance(ANOVA) using JMP Statistical Package (SAS Institute, Cary, N.C.).Significance was determined as p<0.05. Vitals and general laboratorydata are presented as mean±standard error of the mean (sem) unlessotherwise noted.

Calprotectin data were analyzed as follows: entries that were below thelimit of detection (<15 μg/g stool) were converted to 15. Data wereanalyzed by ANOVA (Wilcoxon/Kruskal-Wallis ranked sums test). Data werealso transformed logarithmically to insure homoscendasticity and allstatistical tests were confirmed on the transformed data. Grubb's testfor outliers was performed. Tukey's HSD test was used for post-hoccomparisons.

Results—Subjects—Twenty-six subjects were screened for the trial, and 24subjects were selected for participation (FIG. 1). All subjects enteredthe trial voluntarily and signed informed consents. Of these subjects,12 were assigned to Arm 1, and 11 subjects completed Arm 1. Twelvesubjects were assigned to Arm 2, and 10 subjects completed Arm 2. Threesubjects withdrew from the study, 1 from Arm 1 and 2 from Arm 2. Reasonsfor withdrawal were personal (schedule conflicts and personalinterferences). No adverse events were reported during the trial.

The subjects on Arm 1 ranged in age from 19 years to 44 years, andranged in BMI from 20 to 27 kg/m². The subjects on Arm 2 ranged in agefrom 25 years to 45 years, and ranged in BMI from 24 to 31 kg/m².Subject demographics are summarized in Table 8. TABLE 8 Mean (±sd) age,BMI, and baseline calprotectin for the subjects completing the trial.ARM 1 ARM 2 (N = 11) (N = 10) Age (years) 33 ± 7 33 ± 9 BMI (kg/m²) 22.5± 3.5 27.3 ± 3.0 Calprotectin  44 ± 45  22 ± 24 (μg/g stool)

Clinical observations—No significant changes were observed in vitals (BPor pulse) in subjects on either Arm 1 or Arm 2. BP readings for Arm 1subjects were 125/68±9/8 (N=12) at V1 and 123/68±12/8 (N=10; 1 subject'sdata was not reported) at V6, with no significant changes at the visitin—between V1 and V6. BP readings for Arm 2 subjects were 123/72±7/12(N=12) at V1 and 119/68±7/9 (N=10) at V6, with no significant changes atthe visit in-between V1 and V6. No clinically significant change inweight was noted either, although 5 of the 11 subjects completing Arm 1showed a gain of around 5 lb between V1 and V6 for an average increaseof 3 lb (Table 9). TABLE 9 Mean (±sd) weight for the subjects completingArm 1 (N = 11) and Arm 2 (N = 10) of the trial. Weight (lb) V1 V3 V4 V6Arm 1 168 (25) 168 (24) 169 (24) 171 (25) Arm 2 189 (27) 190 (28) 190(26) 190 (29)

CRP—All subjects—The acute phase reactant, high sensitivity c-reactiveprotein (CRP), has been considered a marker for inflammation. Values ofCRP at <1 mg/L are considered optimal, whereas values of >3.9 mg/L CRPare considered to be elevated. CRP values between 1.1 mg/L and 3.9 mg/Lare considered moderately elevated.

The CRP values at V1 for subjects completing the trial ranged from <0.2mg/L to 2.8 mg/L. Only 7 subjects had values higher than 1 mg/L. Littlechange was seen in CRP values (Table 10) except for 2 subjects on Arm 1,who showed elevated values at V6 only (19.8 mg/L and 23.4 mg/L). TABLE10 Mean (±sd) CRP (mg/L) for the subjects completing Arm 1 (N = 11) andArm 2 (N = 10) of the trial. hs-CRP (mg/L) V1 V3 V4 V6 Arm 1 0.74 (0.82)0.65 (0.44) 0.45 (0.30) 3.19 (6.83) Arm 2 0.87 (0.58) 0.97 (0.66) 1.02(0.96) 2.96 (5.96)

Fecal Calprotectin—Two fecal calprotectin samples were obtained from thesubjects prior to the consumption of the test substances. These baselinesamples were obtained within 4 days of each other. The baseline fecalcalprotectin values are shown in Table 11. Average baseline fecalcalprotectin for all subjects was 37±8 μg/g stool. Ten subjects hadaverage baseline values above 40 μg/g stool, and several subjects hadvalues below the levels of detection (15 μg/g stool). TABLE 11 Baselinecalprotectin baseline data (μg/g stool) for subjects before V1.Independent stool samples taken within 4 days (V1.1 and V1.2) are shown.Data reported as <17 or <16 were converted to 15 (indicating belowdetection). PT# V1.1 V1.2 Ave. Variance 1538 18 18 18 0 1516 36 41 39 51526 48 32 40 16 1527 50 46 48 4 1537 50 44 47 6 1539 58 57 58 1 1520 6139 50 22 1224 212 120 166 92 1521 15 32 23 17 1530 15 15 15 0 1533 15 1515 0 1535 15 15 15 0 1029 33 56 44 12 1532 22 22 22 0 1528 32 34 33 11531 32 19 26 13 1517 69 80 74 11 1536 72 25 48 47 1522 15 15 15 0 152915 15 15 0 1518 15 28 21 13 1525 15 15 15 0 1524 212 ND ND ND 1540 34 2128 13 Average 48 35 41 21 sem 11 5.1 7.1 9.3ND indicates not done.

Fecal calprotectin data for Arm 1 subjects during the trial are shown inTable 12. No significant difference was seen between Baseline 1 (47±13μg/g stool) and V2 and V3 (36±7 μg/g and 36±6 μg/g stool, respectively),when the subjects were consuming IP2003-001. The wash-out value betweenthe cross-over was also consistent with the initial baseline, at 46±14μg/g stool. During the naproxen treatment, however, the fecalcalprotectin value increased to 93±24 μg/g stool and 77±14 μg/g stoolfor V5 and V6, respectively. TABLE 12 Individual calprotectin data (μg/gstool) for subjects completing Arm 1 of the trial. V1 is the average ofthe 2 baseline values. V2 and V3 occurred during treatment withIP2003-001. V4 is the calprotectin value after the 21-day wash-out. V5and V6 occurred during treatment with naproxen. Data reported as <17 or<16 were converted to 15 (indicating the lowest level of detection). PT#V1 V2 V3 V4 V5 V6 1538 18 23 27 25 58 139 1516 39 21 15 15 129 44 152640 54 64 45 81 109 1527 48 39 22 20 22 144 1537 47 26 50 66 87 62 153958 32 15 24 57 62 1520 50 96 60 171 50 64 1224 166 46 51 72 60 115 152123 27 54 42 175 15 1530 15 15 15 15 21 15 1535 15 19 19 66 87 62 AVE 4736 36 46 93 77 sem 13 7.0 6.0 14 24 14

Fecal calprotectin data for Arm 2 subjects during the trial are shown inTable 13. Baseline 1 for these subjects was 27±6.4 μg/g stool. The fecalcalprotectin values during the naproxen treatment, V2 and V3, wereincreased to 93±32 μg/g stool and 96±26 μg/g stool, respectively. Thefecal calprotectin value decreased to approximately Baseline 1 valuesafter the wash-out (32±5.9 μg/g stool), and remained around this valueat V4, during the IP1003-001 treatment (39±7.8 μg/g stool). Someincrease was noted after 14 days on IP2003-001 for these subjects (64±30μg/g stool), although this value was still below those observed duringthe naproxen treatment phase of the trial and was primarily due to 1subject's data (#1517; see FIG. 2). The average fecal calprotectin ofthese subjects at V6 without subject #1417 was 48±17 μg/g stool. TABLE13 Individual calprotectin data (μg/g stool) for subjects completing Arm2 of the trial. V1 is the average of the 2 baseline values. V2 and V3occurred during treatment with naproxen. V4 is the calprotectin valueafter the 21-day wash-out. V5 and V6 occurred during treatment withIP2003-001. Data reported as <17 or <16 were converted to 15 (indicatingbelow detection). PT# V1 V2 V3 V4 V5 V6 1029 44 56 107 25 36 27 1532 2279 77 30 18 36 1528 33 60 78 34 39 57 1531 26 47 48 21 18 15 1517 74 287292 68 90 334 1536 48 274 189 59 42 48 1522 15 17 28 15 15 15 1529 15 3066 15 67 15 1518 21 52 42 41 46 36 1525 15 26 36 15 15 58 AVE 27 93 9632 39 64 sem 6.4 32 26 5.9 7.8 30

Data for each treatment per each Arm were analyzed using paired T-Test(V_(i) with V_(i+1)) and fecal calprotectin values were shown toincrease significantly (p<0.05) for both the 7-day and 14-day naproxentreatment, whereas the IP2003-001 treatment resulted in no significantdifference from Baseline 1. Baseline 1 and Baseline 2 also did notdiffer significantly. Pooled data and are shown graphically in FIGS. 2and 3.

Review of the data showed that some subjects had baseline calprotectinlevels above the reference range. Some literature suggests this couldoccur from temporary upper respiratory infections (URIs), alcoholconsumption, consumption of other drugs that may influence the mucosa,or presence of previously undetected gastrointestinal inflammation(Tibble and Bjarnason, supra, 2001; Meling et al., supra, 1996; Tibbleet al., supra, 1999). Therefore, the data were stratified by thosesubjects with both baselines below the reference range (<50 μg/g stool),and those with at least one baseline above the reference range (=50 μg/gstool).

Of the 21 subjects completing the trial, 15 had both baselines below thereference range. As shown in FIGS. 4 and 5, no significant differencewas seen after 7- and 14-days of IP2003-001, which showed 30±4 μg/gstool and 40±11 μg/g stool, respectively. In this data set, only 1subject had noticeably elevated calprotectin at day 14. In contrast,fecal calprotectin after the naproxen treatment was significantlyelevated (p<0.05) at both 7- and 14-days, at 76±19 μg/g stool and 60±10μg/g stool, respectively. (Note, data are graphed as mean±sd in FIG. 5.)

Six of the 21 subjects completing the trial had at least 1 baselineabove the reference range. In these subjects, the baseline calprotectinlevels (V1 and V4) were 74±19 Og/g stool and 77±20 μg/g stool. As shownin FIGS. 6 and 7, fecal calprotectin after IP2003-001 remainedrelatively stable with baseline in these subjects as well, with 7- and14-day values at 55±12 μg/g stool and 93±49 μg/g stool, respectively.Only 1 subject showed a noticeably elevated calprotectin level at day 4,and this subject's data was the main contributor to the elevation in the14-day calprotectin level after IP2003-001 for this data set. Asobserved with the previous group, however, the fecal calprotectin afternaproxen treatment was elevated at both 7- and 14-days, at 136±46 μg/gstool and 131±38 μg/g stool, respectively, however these changes werenot significant. (Note, data are graphed as mean±sd in FIG. 7.)

Finally, since the 7-day and 14-day treatment data were consistentwithin specific treatments, the 7-day and 14-day data were pooled foranalysis. As shown in Table 14, no difference was noted betweenbaselines. However, fecal calprotectin after the naproxen treatment wassignificantly elevated from baseline (89±11 μg/g stool; p<0.05), whereasthe fecal calprotectin values after IP2003-001 was not significantlyelevated (40±8.2 μg/g stool; ns). TABLE 14 Pooled calprotectin data(mean ± sem) for the subjects on the trial. The number in parenthesesindicates the number of data points per each condition. Baseline 1 isthe average of all V1 baseline values. Baseline 2 is the average of allwash-out values. The 7 day and 14 day data for IP2003-001CT and naproxenwere pooled for this analysis, respectively. Calprotectin Significance(μg/g stool) (p) Baseline 1 (42) 33 ± 5.9 ns Baseline 2 (21) 35 ± 8.5 nsIP2003-001CT (42) 40 ± 8.2 ns Naproxen (42) 89 ± 11* <0.05The * indicates data that are significantly different from Baseline 1 byWilcoxon/Kruskal-Wallis ranked sums.

Summary—A marker of gastrointestinal inflammation, namely assessment byfecal calprotectin, has been used to investigate the effect ofIP2003-001 on gastrointestinal integrity. In this randomized, cross-overstudy, 21 healthy subjects were treated with IP2003-001 (2 tablets bid),or a control substance that has been reported to lead togastrointestinal inflammation and increased fecal calprotectin levels,naproxen (500 mg bid) for 14 days. A 21-day washout occurred between thecrossover treatments and no significant difference in calprotectinlevels was observed between the two baselines. Fecal calprotectinassessment was performed at 7- and 14-days during treatment for each armof the trial and compared to the baselines.

The treatment with IP2003-001 resulted in no significant increase after7 or 14 days. Only 2 subjects showed noticeably elevated calprotectinvalues after the IP2003-001 treatment. In contrast, the naproxentreatment resulted in a noticeable elevation in fecal calprotectin atboth the 7- and 14-day assessments in more than 50% of the subjects.(Note: The increase observed after naproxen is of the same order ofmagnitude as the ˜2-fold increase that has been reported after 7- and14-days of treatment with 500 mg bid; Meling et al., supra, 1996).

Since calprotectin is a marker for gastrointestinal inflammation, thesedata indicate that IP2003-001 leads to little or no gastrointestinalinflammation, as compared to a standard NSAID, and suggest calprotectinmay not carry the risk of ulcers that NSAIDs commonly produce.

EXAMPLE 14 Testing of UltraInflamX™ Anti-Inflammatory Components in theAGS Gastric Mucosal Cell Model

UltraInflamX™ is a low-allergy potential, nutritionally fortified,vegetarian beverage drink mix designed for nutritional support ofchronic inflammatory conditions of the lungs, joints and intestinaltract. UltraInflamX™ can be used as part of a comprehensive eliminationor diet program, as well as nutritional support for patients involved inan anti-inflammatory lifestyle program. As UltraInflamX™ is designed foruse on a chronic basis and several of its components haveanti-inflammatory properties, it was of interest to assess the potentialof these components to induce gastrointestinal toxicity characteristicof non-steroidal anti-inflammatory compounds. The objective of thisstudy was to test the anti-inflammatory components of UltraInflamX™, incombination and individually, for inhibition of PGE₂ biosynthesis in therecently developed AGS human gastric mucosal cell line assay.

The test materials included the active anti-inflammatory components ofUltraInflamX™, curcumin, ginger root, rosemary extract and rutin, and acomposite of these components formulated in the ratio of 2:1:1:2respectively. A minimum of four concentrations, 50, 5, 0.5 and 0.05 μgtest material/mL, with two replicates per concentration over threeindependent experiments were used to compute dose-response curves ofPGE₂ inhibition in AGS cells. The calcium ionophore A23187 was used toinduce arachidonic acid release and was added 60 minutes after exposingthe AGS cells to the test material. Medium inhibitory concentrations(IC_(50s)) with their 95% confidence intervals were computed from theaverage of the three independent experiments. Synergy or antagonism oftest components was quantified using the combination index (CI)parameter. This is discussed in more detail below.

Test Materials and Chemicals—The anti-inflammatory components ofUltraInflamX™, which included curcumin, ginger root, rosemary extractand rutin, were supplied by Metagenics (Gig Harbor, Wash.). A compositeof these materials that was formulated in the ratio present incommercial UltraInflam™ (2:1:1:2 respectively) was also provided byMetagenics.

PGE₂ EIA kits were obtained from Cayman Chemicals (Ann Arbor, Mich.).Heat inactivated Fetal Bovine Serum (FBS-HI Cat. #35-011CV), andDulbecco's Modification of Eagle's Medium (DMEM Cat #10-013CV) waspurchased from Mediatech (Hemdon, Va.). IL-113, aspirin and all standardchemicals, unless noted, were obtained from Sigma (St Louis, Mo.) andwere of the highest purity commercially available. A detailed listing ofsuppliers is presented with each standard operating procedure in theattached appendices.

Cell Culture—The AGS human gastric mucosal cell line (American TypeCulture Collection, Manassas, Va.) was cultured and maintained accordingto recommended ATTC methodology. Subcultured AGS cells were grown inIMDM with 20% FBS, 50 units penicillin/mL, 50 μg streptomycin/mL andmaintained in log phase prior to each experiment. For PGE₂ assays,approximately 10⁵ cells per well were plated into 96-well plates in 200μL growth medium per well. Cells were grown to 80% confluence and washedthree times with IMDA media prior to addition of test agent. Testmaterials were added in 200 μL of IMDA media containing no FBS orpenicillin/streptomycin. Sixty minutes following addition of the testmaterials, arachidonic acid was induced with the addition of the calciumionophore A23187 in DMSO. Incubation at 37° C. was carried out for anadditional 30 minutes. Fifty microliters of media were sampled for PGE₂determination.

Determination of PGE₂—A commercial, non-radioactive procedure forquantification of PGE₂ was employed (Caymen Chemical, Ann Arbor, Mich.)for the determination of PGE₂ and the recommended procedure of themanufacturer will be used without modification. In summary, 50 μL of thesupernatant culture medium, along with a serial dilution of PGE₂standard samples, were mixed with appropriate amounts ofacetylcholinesterase-labeled tracer and PGE₂ antiserum, and incubated atroom temperature for 18 h. Afterwards, the wells in the PGE₂-assaymicrotiter plate were emptied and rinsed with wash buffer, 200 μL ofEllman's reagent containing substrate for acetylcholinesterase were thenadded. The reaction was performed on a slow shaker at room temperaturefor 1 h and the absorbance at 415 nm was determined in a Bio-tekInstruments (Model #Elx800, Winooski, Vt.) ELISA plate reader. Themanufacturer's specifications for this assay include an intra-assaycoefficient of variation of <10%, cross reactivity with PGD₂ andPGF_(2α) of less than 1% and linearity over the range of 10-1000 μgmL⁻¹. The PGE₂ concentration is reported as μg PGE₂/mL.

Cell viability—Cell viability was assessed by visual inspection. None ofthe test materials affected cell viability at the concentrations tested.

Calculations—A minimum of four concentrations, 50, 5, 0.5 and 0.05 μgmaterial/mL, with two replicates per concentration over threeindependent experiments were used to compute dose-response curves andmedium inhibitory concentrations (IC_(50s)) with their 95% confidenceintervals using CalcuSyn (BIOSOFT, Ferguson, Mo.). This statisticalpackage performs multiple drug dose-effect calculations using the MedianEffect methods described by T-C Chou and P. Talalay (Chou, J. Theor.Biol. 35:285-297 (1972); Chou, J. Theor. Biol. 59:253-276 (1976); Chouan Talalay, J. Biol. Chem. 252:6438-6442 (1977); Chou and Talalay, Eur.J. Biochem. 115:207-216 (1981); Chou and Talalay, Adv. Enzymol. 22:27-55(1984)). The fractional inhibition at each dose was averaged over thethree independent experiments and used to calculate dose-response curvesand the median inhibitory concentrations reported. Aspirin was used as apositive control in all experiments. Complete dose-response curves withcoefficients of determination equal to or greater than 0.95 wereobtained for all test materials.

Synergy or antagonism of test components was quantified using thecombination index (CI) parameter. The CI of Chou-Talaly is based on themultiple drug-effect and is derived from enzyme kinetic models (Chou,supra, 1972; Chou, supra, 1976; Cho u and Talalay, supra, 1977; Chou andTalalay, supra, 1981; Chou and Talalay, supra, 1984). The equationdetermines only the additive effect rather than synergism or antagonism.However, synergism is defined as a more than expected additive effect,and antagonism as a less than expected additive effect. Using thedesignation of CI=1 as the additive effect, for mutually exclusivecompounds that have the same mode of action or for mutuallynon-exclusive drugs that have totally independent modes of action thefollowing relationships were obtained: CI<1, =1, and >1 indicatingsynergism, additivity and antagonism, respectively. Additionally, anestimate of the expected median inhibitory concentration of thecomposite was made using the relationship: 1/IC₅₀=A/IC_(50A)+B/IC_(50B)+. . . +N/IC_(50N), where A, B and N were the relative fractions of eachcomponent and A+B+ . . . +N=1.

Results—COX-2 inhibitory activity of UltraInflamX™ components—Medianinhibitory concentrations of PGE₂ synthesis of the test materials in theAGS cell model are presented in Table 15 Aspirin, the positive controlyielded an IC₅₀ of 1.2 μg/mL (95% Confidence Interval=0.37−4.1),consistent with the previously reported value in AGS cells with A23187of 2.9 μg/mL. Of the UltraInflamX™ anti-inflammatory components,curcumin and ginger root exhibited the lowest IC₅₀ values, respectively,2.9 and 4.1 μg/mL. At the other end of the response spectrum, rutin androsemary extract were least inhibitory to PGE₂ synthesis in AGS cellswith IC₅₀ values of 20 and 43 μg/mL, respectively. TABLE 15 Medianinhibitory concentrations (IC50) for PGE₂ synthesis of UltraInflamX ™components in the AGS cell model.† AGS IC₅₀ 95% Confidence IntervalCompound [μM] [μM] r Composite 14 (7.0-30)  0.984 Curcumin 2.9 (1.2-7.2)0.959 Ginger root 4.1 (1.7-10)  0.974 Rosemary extract 43  (8-226) 0.999Rutin 20 (10-22) 0.973 Aspirin - positive control 1.2 (0.37-4.1)  0.950†Values are computed from the average of three independent assays; AGScells were plated and allowed to reach 80% confluence. Cells were washedand test material was added 60 minutes prior to treatment with A23187.Thirty minutes later, media was removed for PGE₂ determination.

Experiments were also performed with a composite of 4 actives. Observedand expected IC₅₀ values for composites was based on weight of wholesample (nine components): observed, 14 μg/mL; expected 11 μg/mL.Observed and estimated IC₅₀ values for composite actives is based uponthe percent weight of the four actives in the composite sample (45.3%):observed, 6.5 μg/mL; expected 5.7 μg/mL.

The composite of anti-inflammatory ingredients produced an IC₅₀ of 14μg/mL. The CI for the composite formulation, computed at 50, 75 and 90percent inhibition were 7.3, 127 and 2250 (Table 16). These high CIvalues indicate extremely strong antagonism among the ingredients. Suchan antagonistic effect is highly desirable, since it indicates adecrease in the expected gastrointestinal toxicity of the compositionrelative to its ingredients. Based upon the IC₅₀ values of eachcomponent and their relative amounts, the expected IC₅₀ of the compositewas estimated at 5.7 μg/mL. This predicted value is below the lowerestimate of the 95% confidence value of 7.0 μg/mL for the composite,supporting the antagonism calculation and CI values (see FIG. 18). TABLE16 Computed combination Index (CI) for the dose-response curvescurcumin, ginger root, rosemary extract, rutin and the UltraInflamX ™composite in a ratio of 2:1:1:2. Test Material CI₅₀ CI₇₅ CI₉₀ Mean CI rUltraInflamX ™ 7.3 127 2250 795 0.984 anti-inflammatory compositeCIs were computed for 50, 75 and 90 percent inhibition of PGE₂biosynthesis in AGS human gastric mucosal cells.

Curcumin with an IC₅₀ of 2.9 μg/mL and ginger root with an IC₅₀ of 4.1μg/mL were similar to aspirin in their inhibitory effects on PGE₂synthesis in the AGS gastric mucosal cell model. Rutin and rosemaryextract were least inhibitory to PGE₂ synthesis with IC₅₀ values of 20and 43 μg/mL, respectively. This combination of ingredients exhibitedexceptionally strong antagonism indicating a decrease in the expectedgastrointestinal toxicity of the composition relative to its components.

As a composite, the anti-inflammatory ingredients of UltraInflamX™, withan AGS IC₅₀ of 14 μg/mL, would be classified as possessing relativelylow gastrotoxic potential. For perspective, this value represents apotential for gastrotoxicity as estimated by the AGS model that is12-times lower than aspirin or 2.5-times lower than rofecoxib.

Based upon the IC₅₀ values of each component and their relative amounts,the expected IC₅₀ of the composite was estimated at 5.7 μg/mL. The CIfor the composite formulation, computed at 50, 75 and 90 percentinhibition were 7.3, 127 and 2250. These high CI values indicateextremely strong antagonism among the ingredients. Such an antagonisticeffect is highly desirable, since it indicates a decrease in theexpected gastrointestinal toxicity of the composition relative to itsingredients.

The components of the test materials are shown in Table 17. TABLE 17Highlighted ingredients in composite test materials. HighlightedIngredient gms/serving Ratio Percent Composite Curcumin† 0.2105 2 15.3Ginger root† 0.1000 1 7.3 Rosemary extract† 0.1000 1 7.3 Rutin† 0.2109 215.4 Quercetin 0.2300 2 16.8 Hespiridin Powder 0.2040 2 14.9 D-Limonene0.1110 1 8.0 N-Acetylcysteine 0.1070 1 7.8 L-Citrulline 0.1000 1 7.3†Potential anti-inflammatory components

The composite sample produced an observed IC₅₀ of 14 μg/mL based upontotal weight of test material, all nine components. This value wasgreater than the expected IC₅₀ for the combination of all ninecomponents estimated at 11 μg/mL. When the IC₅₀ was computed based ononly the four, individually-tested components the observed value was 6.5μg/mL with an expected IC₅₀ of 5.7 μg/mL (see FIG. 19).

The computed combination index (CI) for the composites are shown inTable 18. TABLE 18 Computed combination Index (CI) values for thedose-response curves of the complete composite sample (9 components) andthe four putative, anti-inflammatory components curcumin, ginger root,rosemary extract, and rutin. Test Material CI₅₀ CI₇₅ CI₉₀ Mean CI rUltraInflamX ™ 1.2 22 401 141 0.985 total composite [9] UltraInflamX ™1.1 19 341 120 0.984 anti-inflammatory composite [4]CIs were computed for 50, 75 and 90 percent inhibition of PGE₂biosynthesis in AGS human gastric mucosal cells. CI values for both setsof calculations indicate synergy beginning at the IC₅₀ and increasingdramatically with increasing dose.

Table 19 shows fractional amounts of herbs in a natural product-basedanti-inflammatory. TABLE 19 Fractional amounts of herbs in a naturalproducts based anti-inflammatory. E. DIETARY SUPPLEMENT Amount inRelative (INFLAVONOID IC Formulation Amount INGREDIENTS) [mg][Fraction†] Ascorbic acid (RM07453) 119 0.094 Lemon Bioflavonoid(RM07150) 100 0.079 Boswellin (RM07781) 211 0.166 Cayenne Pepper(RM06572) 25 0.020 Quercetin (RM07671) 57 0.045 Ginger (RM07782) 1000.079 Curcumin Granular (RM06656) 150 0.118 Total of supplements = 7620.601 Complete formulation††† 1267 1.00†Represents supplement fraction of complete formulation (1267 mg)including excipient ingredients listed below.††NA = no activity; parenthetic values are 95% confidence intervals ofthe IC50 calculations.†††Also contained the inert, excipient ingredients dihydrous calciumphosphate (456 mg), magnesium stearate (75 mg), Cabosil (5 mg), Syloid(245 mg) and stearic acid (149 mg).

Spice ingredients from a commercial anti-inflammatory product weretested individually and in combination in a RAW264.7 cell system underconditions assaying COX1 or COX2 activity. As shown in FIG. 20, barswith more gastrotoxic activity (favoring COX1) are to the left of zero,and bars depicting less gastrotoxic (favoring COX2 activity) activityare to the right. These data show that spices such as cayenne pepper,boswellin, and ginger individually have unexpectedly more predicted COX1activity individually than in the fractional combination above(Inflavonoid IC), and demonstrate that combinational effects can existamong various spice ingredients resulting in greater COX2 selectivityand less predicted gastrotoxicity.

Tables 20 and 21 show decreased potential gastropathy of RIAA:rosemarycombinations. TABLE 20 Median inhibitory concentrations (IC₅₀) for PGE₂synthesis of selected natural components in the RAW and AGS cellmodels†. COX-2 RAW COX-2 AGS IC₅₀ Selectivity Compound [μg/mL] [μg/mL]AGS/RAW Curcumin 2.8 2.9 1.0 Ginger root 0.98 4.1 4.2 Rosemary extract0.51 4.0 7.8 RIAA 0.45 21 47 Aspirin - positive control 1.1 0.52 0.48†Values are computed from the average of three independent assays; AGScells were plated and allowed to reach 80% confluence. Cells were washedand test material was added 60 minutes prior to treatment with A23187.Thirty minutes later, media was removed for PGE₂ determination.

TABLE 21 Median inhibitory concentrations (IC₅₀) for PGE₂ synthesis ofselected combinations of RIAA and rosemary in the AGS cell model†. COX-2RAW COX-2 AGS IC₅₀ Selectivity Compound [μg/mL] [μg/mL] AGS/RAW Rosemary0.51 4.0 7.8 RIAA 0.98 21 47 RIAA:Rosemary [2:1]†† 0.61 >50 >82RIAA:Rosemary [1:1]†† 0.67 >50 >74 Aspirin - positive control 1.1 0.520.48†Values are computed from the average of three independent assays; AGScells were plated and allowed to reach 80% confluence. Cells were washedand test material was added 60 minutes prior to treatment with A23187.Thirty minutes later, media was removed for PGE₂ determination.††Estimated from harmonic mean of individual components, assumes nointeraction for RAW cells.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1-16. (canceled)
 17. A method of producing an analgesic and ananti-ulcerogenic effect in a mammal, comprising administering to themammal an amount of a fraction isolated or derived from hops sufficientto produce an analgesic and anti-ulcerogenic effect and a nonsteroidalanti-inflammatory compound, whereby administration of said fractionisolated or derived from hops reduces gastric toxicity associated withsaid non-steroidal anti-inflammatory compound.
 18. The method of claim17, wherein the said fraction isolated or derived from hops comprises acompound of a supragenus having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.
 19. The method of claim 17, wherein saidfraction isolated or derived from hops comprises a compound of Genus Ahaving the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 20. Themethod of claim 17, wherein the fraction isolated or derived from hopscomprises a compound of Genus B having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 21. Themethod of claim 17, wherein said fraction isolated or derived from hopscomprises a compound selected from the group consisting of humulone,cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone.
 22. The method of claim 17, wherein thecomposition comprises about 0.5 to 10000 mg of said fraction isolated orderived from hops.
 23. The method of claim 22, wherein the compositioncomprises about 50 to 7500 mg of the hops derivative.
 24. The method ofclaim 17, wherein the composition comprises about 0.001 to 10 weightpercent of the hops derivative.
 25. The method of claim 24, wherein thecomposition comprises about 0.1 to 1 weight percent of the hopsderivative.
 26. The method of claim 17, wherein the nonsteroidalanti-inflammatory compound is selected from the group consisting ofsalicylic acid, methyl salicylate, difulunisal, salsalate, olsalazine,sulfasalazine, acetanilide, acetaminophen, phenacetin; mefenamic acid,sodium meclofenamate, tolmetin, ketorolac, diclofenac; ibuprofen,naproxen, sodium daproxen, fenoprofen, ketoprofen, flurbioprofen,oxaprozin, piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam,pivoxicam, phenylbutazone, oxyphenbutazone, anitpyrine, aminopyrine,dipyrone; celecoxib, rofecoxib; nabumetone; apazone; nimensulide;indomethacin; sulindac; and etodolac.
 27. The method of claim 26,wherein the nonsteroidal anti-inflammatory compound is selected from thegroup consisting of salicylic acid, methyl salicylate, ibuprofen,naproxen, sodium daproxen, fenoprofen, ketoprofen, flurbioprofen, andoxaprozin.
 28. The method of claim 17, wherein the composition furthercomprises a pharmaceutically acceptable carrier.
 29. The method of claim17, wherein the composition is administered orally, topically,parenterally, or rectally.
 30. The method of claim 17, wherein fractionisolated or derived from hops is administered concomitantly with saidnon-steroidal anti-inflammatory compound.
 31. The method of claim 17,wherein said fraction isolated or derived from hops is administeredafter the administration of said non-steroidal anti-inflammatorycompound.
 32. The method of claim 17, wherein said fraction isolated orderived from hops is administered before the administration of saidnon-steroidal anti-inflammatory compound. 33-36. (canceled)